DBF4 COORDINATES DNA REPLICATION, CHROMOSOME SEGREGATION, AND CHECKPOINT SIGNAL TRANSDUCTION By Ying-Chou Chen A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Genetics - Doctor Of Philosophy 2013 ABSTRACT DBF4 COORDINATES DNA REPLICATION, CHROMOSOME SEGREGATION, AND CHECKPOINT SIGNAL TRANSDUCTION By Ying-Chou Chen Faithful transmission of genetic information not only requires accuracy in DNA synthesis and chromosome segregation, but also surveillance mechanisms that respond to various stresses in order to coordinate repair and cell-cycle progression. Recent evidence suggests that the Dbf4-dependent Cdc7 kinase (DDK), a two-subunit kinase essential for eukaryotic DNA replication, plays such a role in genomic maintenance. In this study, we demonstrated that Dbf4 inhibits Cdc5 (yeast Polo-like kinase) to prevent premature exit from mitosis. It also regulates late origin firing during replication stress by a direct interaction with the Rad53 checkpoint kinase (the ortholog of mammalian Chk2). Dbf4 is able to simultaneously associate with Cdc7, Cdc5, and Rad53, suggesting that Dbf4 serves as a molecular scaffold to mediate DNA replication, chromosome distribution, and checkpoint responses. We further performed a genomewide synthetic lethal screen using a dbf4 mutant, which is defective in binding both Cdc5 and Rad53, to explore the biological relevance of these physical interactions. We globally mapped the genetic interactions of DBF4 and defined the functional categories for these interactions. These data provide insights into the role of Dbf4 in the convergence of checkpoint signaling and mitotic regulation and prompt us to re-evaluate the role of Dbf4 in cell-cycle regulation. ACKNOWLEDGMENTS This dissertation would not have been possible without the guidance of my committee members, help from friends, and support from my family. I would like to gratefully and sincerely thank Dr. Michael Weinreich for his guidance, understanding, and most importantly, his patience during my graduate studies at Van Andel Institute. I appreciate his mentorship not only in directing my projects but also in shaping me into an independent scientist. As growing as a yeast geneticist, Michael provided me many opportunities to attend international meetings where I had the chance to get to know people and researches in my field. These experiences contributed to the development of my long-term career goals. I am very grateful for my dissertation committee, Dr. Min-Hao Kuo, Dr. Steve Triezenberg, and Dr. Steve van Nocker for valuable time and friendship over these years. Additionally, I want to thank our collaborators from the Boone lab at the University of Toronto and the Zegerman lab at the University of Cambridge for technical assistances. I would also like to thank all of the members in the Weinreich lab, especially FuJung Chang and Jessica Kenworthy. FuJung was always willing to help and give me the best suggestions. It would have been lonely in the lab without her. Jessica worked closely with my projects and gave me helpful comments on my manuscripts. It’s been a pleasure to work with all of you. I also need to acknowledge the Van Andel Institute and !!!" the Genetics Program at Michigan State University for being a supportive environment throughout my graduate school years. Finally, I would like to thank my loving friends and family for their full support and encouragement. Without meeting Chin-Mei, Tuan-Mu, and Min-Hao’s family, it would have been a tough winter when I arrived at East Lansing in 2006. Many thanks to Zeynep, Bridget, Jessica, and David for helping my English. Chih-Shia, FuJung, Yi-Mi, Ryan, Wan-Hsuan, Kai-Chun, Joe, Yani, Christine, Emily, Sylvian, Ming-Hui and many friends in Taiwan were also there cheering me up and stood by me through the good times and bad. I would also like to thank my parents and younger sister for their faith in me and allowing me to be as ambitious as I wanted. They were always supporting me and encouraging me with their best wishes. ! iv TABLE OF CONTENTS LIST OF TABLES......................................................................................................... viii LIST OF FIGURES .......................................................................................................... ix KEY TO ABBREVIATIONS........................................................................................... xii CHAPTER 1 BACKGROUND, RATIONALE, AND PURPOSE ........................................................... 1 ABSTRACT...................................................................................................................... 2 INTRODUCTION ............................................................................................................. 2 Cdc7 and Dbf4 are essential for the initiation of DNA replication ................................ 2 Mcm2-7 helicase is the physiological substrate of DDK .............................................. 4 DDK also functions beyond S phase............................................................................ 5 The conserved Dbf4 motif-N is not required for DNA replication ................................. 6 Dbf4 N-terminus interacts with the mitotic Polo-like kinase Cdc5 ................................ 7 THESIS OVERVIEW ....................................................................................................... 9 BIBLIOGRAPHY ............................................................................................................ 12 CHAPTER 2 PART A: CDC7-DBF4 REGULATES MITOTIC EXIT BY INHIBITING POLO KINASE20 ABSTRACT.................................................................................................................... 21 PART B: DBF4 REGULATES THE CDC5 POLO-LIKE KINASE THROUGH A DISTINCT NON-CANONICAL BINDING INTERACTION............................................. 22 ABSTRACT.................................................................................................................... 22 INTRODUCTION ........................................................................................................... 23 RESULTS ...................................................................................................................... 27 Dbf4 residues 82-96 are required to interact with the Cdc5 PBD .............................. 27 A novel binding motif for the Cdc5 PBD ..................................................................... 39 A 14-mer Dbf4 peptide containing residues 83-88 is sufficient for the PBD interaction ................................................................................................................................... 41 Dbf4 uses four key residues to bind the PBD and binding is inhibited by phosphorylation .......................................................................................................... 49 Mutants altering critical residues in the Dbf4 PBD-binding motif suppress the cdc5-1 temperature sensitivity ............................................................................................... 50 Dbf4 inhibits Cdc5 by directly binding the PBD .......................................................... 59 v The PBD interacts with Dbf4 using a surface distinct from its phospho-peptide binding surface ....................................................................................................................... 60 cdc5-HK “pincer” mutant has normal growth rate but shows increased resistance to microtubule disruption ................................................................................................ 66 DISCUSSION ................................................................................................................ 79 An alternative mode of PBD binding .......................................................................... 79 Dbf4 is a scaffold for Cdc5 inhibition .......................................................................... 82 MATERIALS AND METHODS ....................................................................................... 85 ACKNOWLEDGMENTS ................................................................................................ 96 BIBLIOGRAPHY ............................................................................................................ 97 CHAPTER 3 RAD53 BINDS DBF4 THROUGH AN N-TERMINAL T-X-X-E MOTIF AND THIS INTERACTION IS REQUIRED TO SUPPRESS LATE ORIGIN FIRING ................... 104 ABSTRACT.................................................................................................................. 105 INTRODUCTION ......................................................................................................... 106 RESULTS .................................................................................................................... 110 Rad53 interacts with a sequence preceding the Dbf4 BRCT domain ...................... 110 Both Rad53 FHA domains are required to interact with the Dbf4 N-terminus ......... 118 Rad53 FHA domains recognize a T-x-x-E-L motif in the Dbf4 N-terminus .............. 119 The Dbf4-FHA1 domain interaction is phospho-threonine dependent ..................... 131 Dbf4 mediates the association of Cdc7, Rad53, and Cdc5 kinases ........................ 136 A Rad53 checkpoint defect together with loss of specific Dbf4 N-terminal residues results in synthetic lethality ...................................................................................... 137 The Dbf4-Rad53 physical interaction is required to inhibit late origin firing during replication checkpoint activation .............................................................................. 139 DISCUSSION .............................................................................................................. 151 Rad53 interacts with Dbf4 using a phospho-threonine dependent mechanism ....... 151 Models for Rad53 binding to Dbf4............................................................................ 154 FHA1 and FHA2 domains bind to different sites in Dbf4 ...................................... 154 Rad53 FHA domains bind to a Dbf4 dimer using the same sequence ................. 155 Dbf4-Rad53 binding is critical for regulation of late origin activation ....................... 156 Role for a DDK-Rad53-Cdc5 complex? ................................................................... 157 MATERIALS AND METHODS ..................................................................................... 166 ACKNOWLEDGMENTS .............................................................................................. 175 BIBLIOGRAPHY .......................................................................................................... 177 vi CHAPTER 4 FUNCTIONAL CHARACTERIZATION OF THE DBF4 N-TERMINUS BY A GENOMEWIDE SYNTHETIC LETHALITY SCREEN ................................................................ 182 ABSTRACT.................................................................................................................. 183 INTRODUCTION ......................................................................................................... 184 RESULTS .................................................................................................................... 188 An SGA screen for the dbf4-N!109 mutant ............................................................. 188 Dbf4 has strong synthetic interactions with the Top3-Sgs1-Rmi1 complex ............. 194 Genetic and functional interactions between Dbf4 and the CTF complex ............... 200 DDK participates in DNA damage checkpoint response .......................................... 208 Dbf4 genetically interacts with the HIR complex and RNA modulators ................... 209 Dbf4 physically and genetically interacts with the yeast 14-3-3 proteins ................. 210 DISCUSSION .............................................................................................................. 222 Functional characterization of the Dbf4 N-terminus ................................................. 222 It’s all about Rad53 activation .................................................................................. 224 DNA damage response and checkpoint signaling ................................................ 225 Maintenance of replication-fork integrity ............................................................... 227 Rad53-mediated histone homeostasis ................................................................. 229 MATERIALS AND METHODS ..................................................................................... 229 ACKNOWLEDGMENTS .............................................................................................. 242 BIBLIOGRAPHY .......................................................................................................... 243 CHAPTER 5 CONCLUSIONS AND OUTLOOK .............................................................................. 256 RECENT INSIGHTS INTO DDK .................................................................................. 257 Dbf4 is a regulator of chromosome segregation ...................................................... 257 FEAR, MEN, and SPoC........................................................................................ 257 Meiotic recombination and mono-orientation ....................................................... 260 Sister-chromatid cohesion .................................................................................... 261 Dbf4 relays the checkpoint signal ........................................................................... 262 DNA replication checkpoint .................................................................................. 262 DNA damage checkpoint ...................................................................................... 264 Checkpoint adaptation .......................................................................................... 265 CONCLUDING REMARKS .......................................................................................... 267 BIBLIOGRAPHY .......................................................................................................... 270 vii LIST OF TABLES Table 1. Plasmids ......................................................................................................... 89 Table 2. Yeast strains .................................................................................................... 93 Table 3. Peptides ........................................................................................................... 95 Table 4. Plasmids ....................................................................................................... 169 Table 5. Yeast strains .................................................................................................. 173 Table 6. Peptides ......................................................................................................... 174 Table 7. Synthetic lethality or sickness with dbf4-N!109 ........................................... 191 Table 8. Summary of common hits in the SGA screens .............................................. 193 Table 9. Synthetic genetic interaction between dbf4-N!109 and the BLM complex in W303 .......................................................................................................................... 199 ! Table 10. Synthetic genetic interaction between dbf4-N!109 and the CTF complex in W303 .......................................................................................................................... 202 ! Table 11. Validation of the dbf4-N!109 SGA results in W303 ................................... 207 Table 12. Synthetic genetic interaction between dbf4-N!109 and transcriptinal regulators ..................................................................................................................... 214 ! Table 13. Yeast strains ................................................................................................ 233 Table 14. Primers ....................................................................................................... 238 Table 15. Plasmids ..................................................................................................... 241 viii LIST OF FIGURES Figure 1. Mapping the interaction between Dbf4 and the Cdc5 PBD ............................ 28 Figure 2. Analysis of Dbf4 residues required for interaction with the PBD .................... 33 Figure 3. Protein expression of Dbf4 constructs used in two-hybrid and cdc5-1 suppression assays ....................................................................................................... 36 ! Figure 4. Residues required for full length Dbf4 binding to the PBD ............................. 42 Figure 5. A novel, non-consensus polo-box binding sequence in Dbf4 ......................... 45 Figure 6. Dbf4-RSIEGA mutants suppress the cdc5-1 temperature sensitivity ............. 51 Figure 7. Mutations of Dbf4 residues required for the PBD interaction also suppress the cdc5-1 ts ........................................................................................................................ 56 ! Figure 8. Dbf4 binds a surface on the PBD distinct from its phospho-protein binding site ....................................................................................................................................... 61 ! Figure 9. Identification of additional Cdc5 PBD mutations that disrupt the PBD-Spc72 interaction. ..................................................................................................................... 64 ! Figure 10. The Cdc5 pincer residues are not required for yeast viability ...................... 70 Figure 11. Mutation of the Cdc5 pincer residues causes a G2/M delay and alters spindle dynamics........................................................................................................................ 75 ! Figure 12. The dbf4-!82-88 mutant exhibits normal cell cycle progression .................. 78 Figure 13. Mapping the interaction between Dbf4 and Rad53 .................................... 111 Figure 14. Analysis of FHA domain-Dbf4 interactions including a screen of all T/Y residues in Dbf4 residues 100-227 .............................................................................. 115 ! ix Figure 15. The Rad53 FHA domains require a T105-x-x-E-L motif in the Dbf4 N terminus for interaction ............................................................................................................... 121 ! Figure 16. Dbf4 residues V104, T105, E108, L109, and W112 are required for the binding the Rad53 FHA domains ................................................................................. 124 ! Figure 17. The Rad53 FHA1 domain directly binds to a T105 phosphorylated Dbf4 peptide ......................................................................................................................... 127 ! Figure 18. Dbf4 residues V104, E108, and L109 are critical for the specific binding of Rad53 FHA domains ................................................................................................... 133 ! Figure 19. DDK, Rad53 and Cdc5 form a ternary protein complex ............................. 134 Figure 20. dbf4-N!109 is synthetically lethal with rad53-R70A, rad53-K227A, and rad53-G653E ............................................................................................................... 141 ! Figure 21. The synthetic lethality between dbf4-N!109 and rad53-1 or rad53D is not due solely to either loss of Cdc5 interaction or increased Dbf4 stability but requires sequences between residues 82-109 .......................................................................... 144 ! Figure 22. The dbf4-N!109 sld3-38A double mutant allows late origin firing in the presence of HU ............................................................................................................ 146 ! Figure 23. Evidence for a Dbf4-Dbf4 N-terminal interaction ........................................ 160 Figure 24. Sequences between Dbf4 residues 65-88 act to inhibit the Rad53 interaction ..................................................................................................................................... 162 ! Figure 25. Dbf4 T105 residue is critical for the Dbf4-FHA1 domain interaction .......... 164 ! Figure 26. The Dbf4 N-terminus genetically interacts with the Top3-Sgs1-Rmi1 complex ..................................................................................................................................... 198 ! Figure 27. dbf4 ctf double mutants exhibit synthetic defects in growth upon environmental stresses ................................................................................................ 203 ! Figure 28. The Dbf4 N-terminus genetically interacts with Csm1, Pol32, and Rad54 . 205 ! Figure 29. The Dbf4 N-terminus is involved in transcriptional regulation .................... 212 x Figure 30. Mapping the interaction between Dbf4 and yeast 14-3-3 protein ............... 216 ! Figure 31. The Dbf4 N-terminus genetically interacts with Bmh1 ................................ 220 xi KEY TO ABBREVIATIONS 3AT 3 aminotriazole 9-1-1 Rad9-Rad1-Hus1 APC Anaphase Promoting Complex ATM Ataxia Telangiectasia Mutated ATR ATM and Rad3 related BRCT BRCA1 C-Terminal domain CDC Cell Division Cycle CDK Cyclin Dependent Kinase CTF Chromosome Transmission Fidelity C-terminal Carboxy-terminal ! Deletion DDK Dbf4-Dependent Kinase DSB Double Strand Break dsDNA Double Stranded DNA FEAR Fourteen Early Anaphase Release FHA Fork-Head Associated GSA Genetic Synthetic Array GST Glutathione-S-transferase HA HemAgglutinin HU HydroxyUrea xii IP ImmunoPrecipitation MCM MiniChromosome Maintenance MEN Mitotic Exit Network MMS Methyl Methane Sulfonate N-terminal Amino-terminal ORC Origin Recognition Complex PAGE Poly Acrylamide Gel Electrophoresis PBD Polo-Box Domain PCNA Proliferating Cell Nuclear Antigen Plks Polo-like kinases pre-RC pre-Replicative Complex RFC Replication Factor C RPA Replication Protein A Scm Synthetic complete medium Ser Serine SPoC Spindle Position Checkpoint ssDNA single stranded DNA Thr Threonine ts Temperature Sensitive WT Wild Type YPD Yeast extract Peptone Dextrose ! xiii CHAPTER 1 BACKGROUND, RATIONALE, AND PURPOSE 1 BACKGROUND, RATIONALE, AND PURPOSE ABSTRACT The Dbf4-dependent Cdc7 kinase (also known as DDK) is a two-subunit serine/threonine kinase that is essential for the initiation of DNA replication in eukaryotes. The Cdc7 kinase is activated by interacting with the regulatory subunit Dbf4. The expression of Dbf4 oscillates during the cell cycle, analogous to cyclins that activate CDKs (Cyclin-dependent kinases) in a cell-cycle dependent manner. The most characterized function of DDK is to trigger the DNA helicase activity of the Mcm2-7 (Minichromosome maintenance 2-7) complex during S phase. In recent years, accumulating evidence suggests that the Dbf4-dependent Cdc7 kinase not only functions as a DNA replication initiator, but also participates in the regulation of chromosome segregation and the maintenance of genomic integrity. The objective of this study is to determine a novel function of Dbf4 in cell-cycle regulation. INTRODUCTION Cdc7 and Dbf4 are essential for the initiation of DNA replication Chromosome replication in eukaryotic cells is under sophisticated regulation at multiple origins that are distributed throughout the genome (Mechali 2010). In the budding yeast Saccharomyces cerevisiae, the heterohexameric Mcm2-7 DNA helicase, part of the prereplicative complex (pre-RC), is loaded at origins in an inactive form during G1 phase (Bochman and Schwacha 2009). Activation of Mcm2-7 at the onset of S phase depends on both the CDK and Cdc7 kinases. Following the recruitment of other replisome 2 components, active Mcm2-7 complexes unwind the DNA duplex at replication forks to allow for the bidirectional initiation of DNA synthesis. The CDC7 gene was originally identified in Hartwell’s “cell division cycle” mutagenesis screens in the budding yeast Saccharomyces cerevisiae (Culotti and Hartwell 1971), and the dbf4 mutant was characterized as another mutant defective in DNA synthesis which formed a large budded cell called “dumbbell former” morphology at the restrictive temperature (Johnston and Thomas 1982). Later genetic studies showed that the temperature sensitive phenotype of cdc7 mutants was suppressed by overexpression of DBF4 in high copy-number plasmids (Kitada et al. 1992). CDC7 was subsequently found to encode a serine/threonine protein kinase (Hollingsworth and Sclafani 1990), and its kinase activity relied on the presence of Dbf4 during the cell cycle (Johnston and Thomas 1982; Patterson et al. 1986; Yoon and Campbell 1991; Yoon et al. 1993; Oshiro et al. 1999). Studies with budding yeast also showed that both Cdc7 and Dbf4 are needed for the firing of origins throughout S phase (Bousset and Diffley 1998; Donaldson et al. 1998; Tanaka and Nasmyth 1998). Together, these observations suggest that Cdc7 and Dbf4 act in concert to trigger DNA synthesis. In the past decade, it has become clear that Dbf4 directly interacts with and activates Cdc7 to regulate the activation of replication origins; thus Cdc7 kinase is also referred to as Dbf4-dependent kinase (DDK) (Johnston et al. 1999; Sclafani 2000). 3 Mcm2-7 helicase is the physiological substrate of DDK Both DBF4 and CDC7 are essential for viability, but the mcm5-bob1 mutant, which likely mimics the conformational change and activation of Mcm2-7 helicase, is able to suppress the lethality of cdc7! or dbf4! (Hardy et al. 1997; Sclafani 2000; Hoang et al. 2007). This suggests that the Mcm2-7 helicase is the only essential target of DDK. Consistently, DBF4 has been identified as a suppressor of the mcm2-1 mutant in an allele-specific manner (Lei et al. 1997) and physically interacts with Mcm2 (Varrin et al. 2005), one of the subunits in the Mcm2-7 hexamer. Furthermore, five of six conserved Mcm subunits (Mcm2, Mcm3, Mcm4, Mcm6, and Mcm7) were found to be the targets of DDK kinase in vitro and in vivo (Lei et al. 1997; Weinreich and Stillman 1999; Masai et al. 2006; Chuang et al. 2009). However, the biological relevance of DDK-catalyzed Mcm phosphorylation is not completely understood. Based on the studies on the phosphorylation of Mcm4 by DDK (Sheu and Stillman 2006; Sheu and Stillman 2010), it is thought that DDK directly phosphorylates multiple sites on the Mcm2-7 complex and then creates binding sites for other replisome factors, including Cdc45 and the GINS (Sld5-Psf1-Psf2-Psf3, GINS refers to “5-1-2-3” from Japanese “Go-Ichi-Ni-San”) complex. The association of Cdc45 and GINS may drive a structural change in the Mcm2-7 complex and activate its helicase activity (Owens et al. 1997; Weinreich and Stillman 1999; Zou and Stillman 2000; Gambus et al. 2006; Masai et al. 2006; Moyer et al. 2006; Yabuuchi et al. 2006; Francis et al. 2009). 4 DDK also functions beyond S phase The kinase activity of Cdc7 is tightly regulated by the cyclical expression of Dbf4 (Jackson et al. 1993; Johnston et al. 1999). In contrast, Cdc7 protein levels remain constant during the cell cycle. Dbf4 expression is low in G1 phase and reaches a peak at the G1-S transition and throughout the S phase. Notably, Dbf4 is continuously present until late mitosis (Cheng et al. 1999; Weinreich and Stillman 1999; Ferreira et al. 2000; Wu and Lee 2002). Since Cdc7 function is correlated with the presence of Dbf4, this suggests that DDK has additional roles in post-replicative cell cycle regulation. ! Previous studies have shown that Dbf4 stability during the cell cycle is regulated by APC/C (Anaphase-promoting complex or cyclosome) (Cheng et al. 1999; Weinreich and Stillman 1999; Ferreira et al. 2000), which is a large multisubunit E3 ubiquitin ligase that triggers 26S proteasome-mediated degradation of mitotic regulators (Sullivan and Morgan 2007). In particular, APC/C binds to its substrates by recognizing D-box (Destruction-box) and KEN-box sequences through its adapter protein, Cdc20 or Cdh1 (Cdc20-homologue 1) (Pfleger and Kirschner 2000; Pesin and Orr-Weaver 2008). Dbf4 contains two D-boxes in its N-terminus for both Cdc20 and Cdh1 binding (Weinreich and Stillman 1999; Ferreira et al. 2000; Sullivan et al. 2008; Miller et al. 2009; Chen and Weinreich 2010), and it also contains two putative KEN-boxes in the C-terminus that are specifically recognized by Cdh1. It is known that APC/C-Cdc20 is activated at the metaphase/anaphase transition and subsequently targets Dbf4 for degradation. However, Dbf4 was also found in the later mitotic stages, such as during mitotic exit (Sullivan et al. 2008; Miller et al. 2009; Chen and Weinreich 2010), where APC/C-Cdh1 5 plays a dominant role in the regulation of proteolysis. These observations suggest that Dbf4 protein persists late into mitosis and is regulated by both Cdc20 and Cdh1. The regulation of Dbf4 stability could provide insight into the role of Dbf4 in late mitosis. The conserved Dbf4 motif-N is not required for DNA replication A human homolog of Dbf4, called ASK (activator of S-phase kinase), was identified by its association with human Cdc7 in yeast two-hybrid screens (Jiang et al. 1999; Kumagai et al. 1999). A second DBF4-like gene was found in the mammalian genome called DRF1 (Dbf4-related factor 1) /DBF4B/ASKL1 (ASK-like protein 1) (YoshizawaSugata et al. 2005), but the DRF1 homolog was not found in S. cerevisiae or S. pombe. The Cdc7 kinase and the regulatory subunit of Dbf4 are both evolutionarily conserved, but they show different degrees of sequence conservation from yeast to humans (Johnston et al. 2000). All Cdc7 homologs share well-conserved residues for serine/threonine kinase activity, but much less conservation is observed in its binding partner Dbf4, which contains no catalytic domain. Sequence alignment among S. cerevisiae Dbf4, S. pombe Dbf4/dfp1+, and human ASK shows three short conserved motifs, termed motif-N (N-terminal), -M (Middle), and -C (C-terminal) (Masai and Arai 2000). The most conserved motif-M and zinc-finger motif-C of Dbf4/Dfp1 are sufficient to bind and activate the Cdc7/Hsk (in S. pombe) kinases in yeasts and thus are essential for viability (Ogino et al. 2001; Harkins et al. 2009; Jones et al. 2010). In contrast, motif-N is dispensable for viability, but could be involved in targeting DDK to stalled replication forks during replication stress (Ogino et al. 2001; Duncker et al. 2002; Gabrielse et al. 2006). Deletion of the Dbf4 N-terminus causes increased sensitivity to 6 DNA-damaging agents such as hydroxyurea (HU), ultraviolet (UV) light, and methyl methanesulfonate (MMS). These observations suggest that the Dbf4 N-terminus plays a role in cellular response to environmental stresses. ! The Dbf4 N-terminus has been reported to contain a BRCT (BRCA1 C-terminal)-like domain, which generally forms tandem repeats and functions as a phospho-peptide binding module in signal transduction (Gabrielse et al. 2006; Mohammad and Yaffe 2009; Matthews et al. 2012). The BRCT domain is often found in proteins involved in checkpoint response and DNA repair. It was been shown that the BRCT domain of Dbf4 interacts with the Rad53 checkpoint kinase, the ortholog of the human tumor suppressor Chk2 (Checkpoint kinase 2) (Duncker et al. 2002; Chen et al. 2012; Matthews et al. 2012). During replication stress, Rad53 activation coincides with the hyperphosphorylation of Dbf4 and subsequently attenuates DDK kinase activity at late replication origins (Gabrielse et al. 2006; Yabuuchi et al. 2006; Lopez-Mosqueda et al. 2010; Zegerman and Diffley 2010; Chen et al. 2012). An attractive model is that the Dbf4 N-terminus is a direct target of the Rad53 kinase during S-phase checkpoint signaling and that the phosphorylated Dbf4 prevents the replicative function of Cdc7, allowing DNA repair to take place before further S phase progression.! ! Dbf4 N-terminus interacts with the mitotic Polo-like kinase Cdc5 ! Earlier reports have shown that Dbf4 N-terminus physically associates with the Cdc5 kinase (Hardy and Pautz 1996; Miller et al. 2009; Chen and Weinreich 2010), a homolog of mammalian Polo-like kinases (Plks). The Polo gene was named for a 7 Drosophila melanogaster mutant that exhibited aberrant mitotic spindle and spindle pole bodies (equivalent to the centrosomes in higher eukaryotes), implying that Polo had a crucial role in chromosome segregation (Sunkel and Glover 1988; Barr et al. 2004; Archambault and Glover 2009). Polo kinases are now known to form a large protein family, and they regulate centrosome maturation and duplication, mitotic entry, chromosome segregation, spindle dynamics, and mitotic exit (Lee et al. 2005). Budding yeast (CDC5), fission yeast (plo1+), and Drosophila (Polo) each have a single Polo ortholog, but mammalian cells have four Polo-like kinases (Plk1, Plk2, Plk3, and Plk4/SAK) (Dai 2005). The function of Cdc5 closely resembles the human Plk1 (Lee and Erikson 1997; Ouyang et al. 1997), which is frequently associated with malignant types of cancers (Eckerdt et al. 2005; Takai et al. 2005). Different molecular compounds are being developed to inhibit Plk1 kinase activity and its non-catalytic substrate binding domain (Strebhardt and Ullrich 2006; de Carcer et al. 2007; Reindl et al. 2008; Watanabe et al. 2009). ! Like the other Polo family members, Cdc5 plays multiple essential roles in mitosis and meiosis. Cdc5 is required for the phosphorylation of Swe1 (Saccharomyces Wee1), which is a negative regulator of S-phase CDKs at the G2/M transition (Bartholomew et al. 2001; van Vugt and Medema 2004; Asano et al. 2005). A deficiency in Cdc5mediated Swe1 phosphorylation delays mitotic entry. Similarly, Cdc5-mediated phosphorylation of cohesins (Mcd1/Scc1; Mitotic chromosome determinant) is crucial in triggering the onset of the metaphase to anaphase transition (Alexandru et al. 2001; Hornig and Uhlmann 2004). In later stages of mitosis, Cdc5 regulates the FEAR (Cdc 8 fourteen early anaphase release) network and MEN (Mitotic exit network) to facilitate the activation of Cdc14 (Lee et al. 2001; Shou et al. 2002; Stegmeier et al. 2002; Visintin et al. 2003; Rahal and Amon 2008), which is an essential phosphatase that antagonizes mitotic CDK functions during mitotic exit (Stegmeier and Amon 2004). Cdc5 is also implicated in signal transduction during cytokinesis (Valentin et al. 2006; Katis et al. 2010). Intriguingly, recent studies demonstrated that DDK controls meiosisspecific transcription (Lo et al. 2008; Lo et al. 2012), the separase-mediated cleavage of Rec8 (a meiotic cohesin component) (Valentin et al. 2006; Katis et al. 2010), and the disjunction of homologous chromosomes at meiosis I in budding yeast (Valentin et al. 2006; Matos et al. 2008; Wan et al. 2008), suggesting that DDK has additional roles in orchestrating meiotic events. Therefore, the physical interaction between Dbf4 and Cdc5 raises interesting questions regarding whether Dbf4 and Cdc7 participate in mitotic or meiotic events through the interaction with Cdc5. THESIS OVERVIEW ! Saccharomyces cerevisiae DBF4 encodes an essential regulator for the S-phase kinase Cdc7. While the function of Dbf4 in DNA replication has been studied over the past decade, the role of Dbf4 in post-replicative cell-cycle regulation is less understood, and will be the focus of this study. Genetic and biochemical observations suggest that the non-essential N-terminus of Dbf4 interacts with the mitotic Polo-like kinase Cdc5 and checkpoint kinase Rad53. We hypothesized that Dbf4 participates in the post-replicative cell-cycle regulation through direct interactions with Cdc5 and Rad53, and aimed to 9 identify the molecular mechanism of Dbf4 function in mitotic regulation and checkpoint signal transduction. In Chapter 2 and 3, we characterized two distinct motifs within the Dbf4 N-terminus that physically interact with Cdc5 and Rad53, separately. By examining the dbf4 mutants that are unable to interact with Cdc5 or Rad53, we elucidated the novel roles of Dbf4 in the mitotic exit network and the regulation of origin firing during replication stress. Furthermore, we found that Dbf4 simultaneously forms a stable complex with Cdc7, Cdc5 and Rad53 kinases, suggesting that Dbf4 functions as a scaffold that coordinates DNA replication, chromosome segregation and checkpoint signaling during cell cycle. In Chapter 4, we performed a genome-wide synthetic lethal screen by using a dbf4 mutant, which is defective in binding both Rad53 and Cdc5, to further explore the biological relevance of physical interactions. We globally mapped the genetic interactions of DBF4 and defined the functional categories for these interactions, including the maintenance of genomic stability, DNA damage or checkpoint signaling, and chromosome segregation. These data suggest that Dbf4, Cdc5, and Rad53 operate in parallel pathways to repair damaged or stalled forks and also to block inappropriate mitotic progression in response to damaged or partially replicated chromosomes.! ! In Chapter 5, we review recent studies on the molecular basis of Dbf4-Cdc5 and Dbf4Rad53 interactions and also discuss the potential role of ternary complex (Dbf4-Cdc7Cdc5-Rad53) outside the S phase. Taken together with the data of synthetic lethal screen, our results contribute to a comprehensive understanding of Dbf4 function in cell- 10 cycle regulation and we propose a model in which Dbf4 acts as a multifaceted cell-cycle regulator. 11 BIBLIOGRAPHY 12 BIBLIOGRAPHY Alexandru, G., Uhlmann, F., Mechtler, K., Poupart, M.A., and Nasmyth, K. 2001. Phosphorylation of the cohesin subunit Scc1 by Polo/Cdc5 kinase regulates sister chromatid separation in yeast. Cell 105(4): 459-472. Archambault, V. and Glover, D.M. 2009. Polo-like kinases: conservation and divergence in their functions and regulation. Nat Rev Mol Cell Biol 10(4): 265-275. Asano, S., Park, J.E., Sakchaisri, K., Yu, L.R., Song, S., Supavilai, P., Veenstra, T.D., and Lee, K.S. 2005. 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Yoshizawa-Sugata, N., Ishii, A., Taniyama, C., Matsui, E., Arai, K., and Masai, H. 2005. A second human Dbf4/ASK-related protein, Drf1/ASKL1, is required for efficient progression of S and M phases. J Biol Chem 280(13): 13062-13070. Zegerman, P. and Diffley, J.F. 2010. Checkpoint-dependent inhibition of DNA replication initiation by Sld3 and Dbf4 phosphorylation. Nature 467(7314): 474-478. Zou, L. and Stillman, B. 2000. Assembly of a complex containing Cdc45p, replication protein A, and Mcm2p at replication origins controlled by S-phase cyclindependent kinases and Cdc7p-Dbf4p kinase. Mol Cell Biol 20(9): 3086-3096. 19 CHAPTER 2 PART A: CDC7-DBF4 REGULATES MITOTIC EXIT BY INHIBITING POLO KINASE Charles T. Miller, Carrie Gabrielse, Ying-Chou Chen, and Michael Weinreich Part A was published by PLoS Genetics, 5(5):e1000498 (2009). Ying-Chou Chen identified that the Dbf4 residues 82-88 are necessary for the interaction with Cdc5 and also demonstrated that the Dbf4-mediated Cdc5 inhibition depends on the association of Cdc7. These discovery contributed to Figure 1A, 1B, 3A, 4A, 4B, and 4C in this paper. PART B: DBF4 REGULATES THE CDC5 POLO-LIKE KINASE THROUGH A DISTINCT NON-CANONICAL BINDING INTERACTION Ying-Chou Chen and Michael Weinreich Part B was published by Journal of Biological Chemistry, 31;285(53):41244-54 (2010). 20 PART A: CDC7-DBF4 REGULATES MITOTIC EXIT BY INHIBITING POLO KINASE ABSTRACT Cdc7-Dbf4 is a conserved protein kinase required for the initiation of DNA replication. The Dbf4 regulatory subunit binds Cdc7 and is essential for Cdc7 kinase activation, however the N-terminal third of Dbf4 is dispensable for its essential replication activities. Here we define a short N-terminal Dbf4 region that targets Cdc7-Dbf4 kinase to Cdc5, the single Polo kinase in budding yeast that regulates mitotic progression and cytokinesis. Dbf4 mediates an interaction with the Polo substrate-binding domain to inhibit its essential role during mitosis. Although Dbf4 does not inhibit Polo kinase activity it nonetheless inhibits Polo-mediated activation of the mitotic exit network (MEN), presumably by altering Polo substrate targeting. In addition, although dbf4 mutants defective for interaction with Polo transit S-phase normally, they aberrantly segregate chromosomes following nuclear misorientation. Therefore, Cdc7-Dbf4 prevents inappropriate exit from mitosis by inhibiting Polo kinase and functions in the spindle position checkpoint. 21 PART B: DBF4 REGULATES THE CDC5 POLO-LIKE KINASE THROUGH A DISTINCT NON- CANONICAL BINDING INTERACTION ABSTRACT Cdc7-Dbf4 is a conserved, two-subunit kinase required for initiating eukaryotic DNA replication. Recent studies have shown that Cdc7-Dbf4 also regulates the mitotic exit network (MEN) and monopolar homolog orientation in meiosis I. Both activities likely involve a Cdc7-Dbf4 interaction with Cdc5, the single Polo-like kinase in budding yeast. We previously showed that Dbf4 binds the Cdc5 polo-box domain (PBD) via a ~40 residue N-terminal sequence, which lacks a PBD consensus binding site (S(pS/pT)P/X), and that Dbf4 inhibits Cdc5 function during mitosis. Here we identify a non-consensus PBD binding site within Dbf4 and demonstrate that the PBD-Dbf4 interaction occurs via a distinct PBD surface from that used to bind phospho-proteins. Genetic and biochemical analysis of multiple dbf4 mutants indicate that Dbf4 inhibits Cdc5 function through direct binding. Surprisingly, mutation of invariant Cdc5 residues required for binding phosphorylated substrates has little effect on yeast viability or growth rate. Instead, cdc5 mutants defective for binding phospho-proteins exhibit enhanced resistance to microtubule disruption and an increased rate of spindle elongation. This study therefore details the molecular nature of a new type of PBD binding and reveals that Cdc5 targeting to phosphorylated substrates likely regulates spindle dynamics. 22 INTRODUCTION Cell cycle progression requires the highly accurate replication and segregation of chromosomes. Although these two events occur at different times, several cell cycle kinases regulate both DNA synthesis and chromosome segregation (Blow and Tanaka 2005). In budding yeast, the Cdc7-Dbf4 kinase (also called Dbf4-dependent kinase or DDK) plays such a dual role in the cell cycle. The Dbf4 regulatory subunit binds to and activates Cdc7 kinase to initiate DNA replication (Johnston et al. 1999; Jares et al. 2000). DDK also promotes other aspects of chromosome biology including cohesin loading during early S-phase in X. laevis (Takahashi et al. 2008), centromeric cohesion in S. pombe (Takahashi et al. 2008), and meiotic recombination (Sasanuma et al. 2008; Wan et al. 2008) and the Ndt80 (early meiotic) transcriptional program in S. cerevisiae (Lo et al. 2008). Budding yeast DDK also promotes monopolar orientation of homologs in meiosis I and inhibits chromosome segregation in the mitotic cycle (Matos et al. 2008; Sullivan et al. 2008; Marston 2009; Miller et al. 2009). Both activities are likely mediated through an interaction with Cdc5, the single Polo-like kinase in Saccharomyces cerevisiae. Polo-like kinases (Plks) regulate mitotic events and are also involved in the response to DNA damage and checkpoint adaptation (Lee et al. 2005; Petronczki et al. 2008; Trenz et al. 2008). Genetic and physical interactions between Dbf4 and Cdc5 were described many years ago (Kitada et al. 1993; Hardy and Pautz 1996) raising the possibility that DDK acted beyond S phase. The DDK-Cdc5 interaction raises interesting questions regarding how these distinct kinases interact and coordinate accurate cell cycle progression. 23 The Polo gene was named for a Drosophila melanogaster mutant that exhibited abnormal spindle pole behavior (Sunkel and Glover 1988), implying that Polo had a critical role in mitotic organization. Polo kinases are now known to comprise a large protein family that regulate centrosome maturation and duplication, mitotic entry, chromosome segregation, spindle dynamics, and mitotic exit (Archambault and Glover 2009). Budding yeast, fission yeast and Drosophila each have a single Polo ortholog but there are four Polo-like kinases (Plk1-4) in mammalian cells (Archambault and Glover 2009). Consistent with Polo’s diverse functions, individual Plks show different and sometimes dynamic subcellular localization (Barr et al. 2004). Polo kinases share a two-domain structure consisting of an N-terminal kinase domain and a C-terminal substrate-binding domain. A unique C-terminal Polo-box domain (PBD) comprised of one or two polo-box (PB) motifs was found in all Polo family members by multiple sequence alignment (Lowery et al. 2005), and is required for Plk subcellular localization and substrate targeting (Lee et al. 1998; Seong et al. 2002; Lee et al. 2005). The PBD is one of many domains that bind phosphorylated substrates (Yaffe and Smerdon 2004). The interaction between an optimal phospho-threonine peptide and the PBD of Plk1 has been defined by structural and mutational studies (Cheng et al. 2003; Elia et al. 2003b). The polo-box domains of Plk1-3 orthologs are constituted from two highly conserved polo-box sequences, called PB1 and PB2, together with a polo cap (Pc) region that stabilizes the folded domain. Over 600 Plk substrates were suggested in proteomic study using the phosphorylation-recognition feature of the PBD (Lowery et al. 2007) suggesting that Plks regulate many substrates. Since Plk1 overexpression occurs in human tumors, Polo kinases are attractive targets for cancer therapy (Strebhardt and 24 Ullrich 2006). In fact, different molecular approaches are being developed to inhibit both Plk1 kinase activity and its noncatalytic substrate-binding domain (Strebhardt and Ullrich 2006; de Carcer et al. 2007; Reindl et al. 2008; Watanabe et al. 2009). The CDC5 gene was first described in a cell division cycle mutant screen by Hartwell and colleagues through the isolation of a single cdc5-1 temperature sensitive allele (Hartwell et al. 1970). Like the other Polo family members, Cdc5 has multiple roles in mitosis and cytokinesis (Lee et al. 2005). Human Plk1 can complement the growth defect of the yeast cdc5-1 mutant, which provided further evidence that Polo functional interactions were conserved during evolution (Lee and Erikson 1997; Ouyang et al. 1997). Despite a broad spectrum of potential Cdc5 substrates, only a few PBD-binding interactions have been characterized in detail (Geymonat et al. 2003; Hornig and Uhlmann 2004; Lowery et al. 2004; Asano et al. 2005; Snead et al. 2007; Crasta et al. 2008). We recently performed a two-hybrid screen using the Dbf4 N-terminus and defined a Dbf4 interaction with the Cdc5 PBD (Miller et al. 2009). We further found that Dbf4 residues 66-109 were necessary and sufficient for this interaction. However, this Dbf4 region did not contain a recognizable PBD consensus binding sequence, i.e. SerpSer/pThr-Pro/X (“p” denotes phosphorylation and “X” indicates any amino acid), and mediated an interaction with the PBD without a requirement for phosphorylation. Similarly, Glover and colleagues reported that the PBD of Drosophila Polo mediates an interaction with Map205 (a microtubule-associated protein) that occurs in the absence of Map205 phosphorylation (Archambault et al. 2008). 25 Here we systematically map Dbf4 residues required for binding the PBD using genetic and direct peptide binding assays. Although targeted deletion of Dbf4 residues 83-88 or 89-93 completely abrogates Dbf4-Cdc5 binding in vivo, only residues 83-88 are critical for a direct PBD interaction and comprise the core of a new type of PBD binding sequence. Furthermore, the PBD interacts with Dbf4 independently of residues that mediate its interaction with phosphorylated proteins using a distinct molecular surface. Surprisingly, highly conserved Cdc5 residues (W517, H641, K643) in the PBD, required for binding proteins with an S(pS/pT)P/X consensus sequence, are not required for yeast viability or wild-type growth rates. This strongly suggests that Cdc5 binding to phosphorylated (primed) substrates is not essential in yeast. Instead, the cdc5-HK and cdc5-WHK mutants exhibit enhanced resistance to spindle poisons and display altered spindle dynamics. These data define an alternative mode for PBD-protein interactions, and raise the possibility that Cdc5 may bind essential mitotic substrates through a Dbf4like consensus sequence. 26 RESULTS Dbf4 residues 82-96 are required to interact with the Cdc5 PBD We previously recovered multiple clones of the Cdc5 PBD in a two-hybrid screen using the Dbf4 N-terminus as bait and found that residues 66-109 are necessary and sufficient for a direct interaction with Cdc5 PBD (Miller et al. 2009). Since Dbf4 Nterminal residues 1-109 are dispensable for DNA replication (Gabrielse et al. 2006), the Dbf4 N-terminus interacts with Cdc5 to perform non-essential functions in budding yeast. To define the exact molecular basis of the Dbf4-Cdc5 interaction, we constructed a series of N-terminal Dbf4 deletion mutants and tested their ability to interact with the Cdc5 PBD using a two-hybrid assay. Deletions to residue 82 did not significantly affect the PBD two-hybrid interaction, however, N-terminal deletions extending beyond residue 82 lost the ability to interact with the PBD (Figure 1A, B). We then truncated the C-terminus and found that Dbf4 residues 66-96 were sufficient for PBD binding. Deletion of residues 82-88 (as shown previously (Miller et al. 2009)), 89-93, or 82-96 eliminated PBD binding (Figure 1A, B). This data indicated that sequences between residues 82 and 96 were essential for the Dbf4-Cdc5 interaction but did not define which residues directly contact the PBD. 27 Figure 1. Mapping the interaction between Dbf4 and the Cdc5 PBD (A) N-terminal Dbf4 deletion mutants were tested for a two-hybrid interaction with the PBD. 10-fold serial dilutions of saturated cultures were spotted onto SCM-Trp-Leu plates to visualize total cells and SCM-Trp-Leu-His + 2 mM 3AT plates, to score the two-hybrid interaction. (B) Schematic of the features in Dbf4 N-terminus are shown, including two potential destruction-boxes (D-boxes), a conserved BRCT-like domain and motifs N, M and C, along with a summary of the Dbf4PBD two-hybrid data. (C) Two-hybrid results for various point mutants spanning Dbf4 residues 82-96 are summarized. R83, I85, G87, and A88 are critical for PBD binding. (D) HA-Cdc7-Dbf4 complexes were immunoprecipitated from baculovirus-infected Sf9 cells and examined for co-immunoprecipitation of 3Myc-Cdc5. Cdc5 was co- immunoprecipitated by wild-type Dbf4 but not by Dbf4-!82-88 and Dbf4-N!109 mutant proteins. 28 Figure 1. (cont’d) 29 Figure 1. (cont’d) 30 Figure 1. (cont’d) 31 Figure 1. (cont’d) 32 Figure 2. Analysis of Dbf4 residues required for interaction with the PBD (A) The indicated Dbf4 (66-227) bait constructs were assayed for a two hybrid interaction with the Cdc5 PBD by spotting serial dilutions of cultures onto the indicated media to visualize the total number of cells (left) and the two hybrid interaction (right). (B) Although deletion of Dbf4 residues 89-93 abolishes the PBD interaction, deletion of residues 89-91 (!VQV) has only a modest effect on the PBD interaction and deletion of residues 92-93 (!SK) has no effect. This strongly suggests that deletion of residues 8993 indirectly affects the Dbf4-PBD interaction. (C) The VQV89AAA triple point mutant has a similar effect on the PBD interaction as deletion of these same residues, as shown in panel B. However, the V89A, Q90A, and V91A single mutants have no effect on the PBD interaction. 33 Figure 2. (cont’d) 34 Figure 2. (cont’d) 35 Figure 3. Protein expression of Dbf4 constructs used in two-hybrid and cdc5-1 suppression assays (A) The protein expression level of selected Gal4 DNA binding domain (DB) fusions to Dbf4(66-227) and representative point mutants spanning residues 82-93 were visualized by immunoblotting. Ponceau S staining (left) of whole cell extracts verified equal loading in each lane. Dbf4 bait constructs (DBD-Dbf4) contained a Myc tag and were detected using antiMyc antibody (9E10). (B) Protein expression level of full length Dbf4 wild type and critical point mutants expressed in M2600 (dbf4!::kanMX6 cdc5-1). 36 Figure 3. (cont’d) 37 Figure 3. (cont’d) 38 A novel binding motif for the Cdc5 PBD Cdc5 is most closely related to the Plk1 family and its C-terminal PBD shares about 36% identity with the PBD of human Plk1 (Lee et al. 2005). Using an oriented peptidelibrary screen, the PBD of Cdc5 and Plk1 were found to preferentially bind SerpSer/pThr-Pro/X peptides (Elia et al. 2003b). For both Plk1 and Cdc5, the serine preceding the phosphorylated residue is absolutely required for PBD binding in vitro (Elia et al. 2003a; Elia et al. 2003b). The current model for Polo targeting suggests that a priming kinase, such as a cyclin-dependent kinase (Cdk) or MAP kinase, phosphorylates selected Ser/Thr residues on Polo substrates to create a high-affinity PBD recognition motif (Elia et al. 2003b; Barr et al. 2004). Plk1 also uses self-priming to create its own high-affinity binding site on PBIPB1 (Kang et al. 2006). However, several Cdc5 or Plk substrates apparently do not require the priming kinases for PBD binding (Garcia-Alvarez et al. 2007; Archambault et al. 2008; Rahal and Amon 2008) and Dbf4 residues 82-96 do not contain a match to the PBD consensus-binding site. In order to determine individual Dbf4 residues required for PBD binding, we constructed a series of point mutants spanning residues 82-96 and quantitated the Dbf4 two-hybrid interaction with the PBD. Mutations of residues R83, I85, G87 and A88 completely abrogated the interaction with the PBD similar to deletion of residues 82-88 (Figure 1C; see Figure 2 for two-hybrid spotting data), although the mutant proteins were expressed similarly to the wild type (Figure 3, and data not shown). In contrast, the A82V, S84A, S84E, E86A, and E86K mutations had little effect on the Dbf4-PBD interaction (Figure 1C). Although the VQV89-91AAA mutation had a modest effect on PBD binding, the 39 SK92,93AA and GTG94-96AAA mutations had no effect (Figure 1C). The V89A, Q90A, and V91A single point mutations also had no effect on the Dbf4-PBD interaction (Figure 2). Together, these observations suggest that Dbf4 residues 83-88 directly bind the Cdc5 PBD in a phosphorylation-independent manner. Since deletion of residues 89-93 eliminated the PBD interaction but mutation of individual amino acids within this sequence had no effect on binding, it is likely that residues 89-93 do not directly contact the PBD or they make non-essential contacts. Based on this point mutant analysis we suggest that Dbf4 residues 83-RSIEGA-88 comprise the core of a novel PBD binding motif. Lastly, we tested whether residues 82 and following were required for PBD binding in the context of full length Dbf4. Although full length Dbf4 interacted with the PBD, dbf4 mutants deleting past residue 82 failed to interact (Figure 4) indicating that these residues were critical for the interaction in full length Dbf4. To examine Dbf4-Cdc5 in the context of functional Cdc7-Dbf4 kinase (DDK), we tested the ability of wild type Dbf4 and Dbf4-!82-88 proteins to co-immunoprecipitate Cdc5 using a baculovirus expression system. HA-Cdc7, Dbf4 and Myc-Cdc5 proteins were co-expressed in Sf9 cells and the HA-Cdc7-Dbf4 complex was immunoprecipitated using an anti-HA monoclonal antibody. Cdc5 was co-immunoprecipitated by DDK complexes containing wild type Dbf4, but not by DDK complexes containing the Dbf4-!82-88 and Dbf4-N!109 mutant proteins (Figure 1D). These data indicate the DDK-Cdc5 interaction requires Dbf4 residues 8288. 40 A 14-mer Dbf4 peptide containing residues 83-88 is sufficient for the PBD interaction We next defined the minimal Dbf4-interacting peptide using the two-hybrid assay. A short Dbf4 peptide containing only residues 78-96 was sufficient for PBD-binding (Figure 5A). Although residues 82-96 did not bind the PBD, this was likely due to assay constraints and not due to loss of critical residues from 78-81, i.e. a quadruple alanine mutant of residues 78-81 (“RIER” to “AAAA”) bound the PBD as well as wild-type Dbf4 66-96 (Figure 5A). This demonstrates that a 19 amino acid peptide (78-96) containing the Dbf4 sequence 83-RSIEGA-88 is sufficient for PBD binding. To verify that Dbf4 residues 82-88 comprise a unique PBD binding motif, we first tested the ability of Dbf4 peptides to directly interact with the purified Cdc5 PBD using the AlphaScreen assay (Ullman et al. 1994). In this assay, a biotinylated Dbf4 peptide and purified His6-PBD are bound to streptavidin (donor) and Ni++ (acceptor) beads, respectively. Excitation with 680 nm light causes donor beads to emit singlet oxygen, which activates fluorophores in proximally bound acceptor beads to emit light at 520620 nm. A biotinylated Dbf4 peptide (73-96), but not an unrelated peptide, interacted with purified Cdc5 polo-box domain (residues 357-705) in a dose dependent manner (Figure 5B). 41 Figure 4. Residues required for full length Dbf4 binding to the PBD (A) Two hybrid assays indicate that deletion of residues 82-88 within full-length Dbf4 completely disrupts the Dbf4-PBD interaction. Although deletion of N-terminal 65 residues did not affect the PBD two-hybrid interaction, the interaction was lost by the addition of the 82-88 deletion. (B) Diagram of full-length (FL) Dbf4 constructs used in two-hybrid assays. The dbf4-N!65 mutant disrupts two destruction boxes at residues 10-19 and 62-70. 42 Figure 4. (cont’d) 43 Figure 4. (cont’d) 44 Figure 5. Analysis of Dbf4 residues required for interaction with the PBD (A) The indicated Dbf4(66-227) bait constructs were assayed for a two hybrid interaction with the Cdc5 PBD by spotting serial dilutions of cultures onto the indicated media to visualize the total number of cells (left) and the two hybrid interaction (right). (B) Although deletion of Dbf4 residues 89-93 abolishes the PBD interaction, deletion of residues 89-91 (!VQV) has only a modest effect on the PBD interaction and deletion of residues 92-93 (!SK) has no effect. This strongly suggests that deletion of residues 89-93 indirectly affects the Dbf4-PBD interaction. (C) The VQV89AAA triple point mutant has a similar effect on the PBD interaction as deletion of these same residues, as shown in panel B. However, the V89A, Q90A, and V91A single mutants have no effect on the PBD interaction. 45 Figure 5. (cont’d) 46 Figure 5. Analysis of Dbf4 residues required for interaction with the PBD 47 Figure 5. (cont’d) 48 To confirm the specificity of the binding assays, non-biotinylated Dbf4 peptides of differing lengths but containing the Dbf4-RSIEGA sequence were tested for their ability to compete the biotinylated-peptide PBD interaction. Four different peptides ranging from 24 to 14 residues showed a similar ability to compete the Dbf4-PBD interaction (Figure 5C), strongly suggesting that the RSIEGA residues directly bind the PBD. The affinity of Dbf4 peptide binding to the PBD in this assay was 1-5 µM as determined by competition with an unlabeled Dbf4 peptide (Figure 5C). Dbf4 uses four key residues to bind the PBD and binding is inhibited by phosphorylation We used peptide competition assays to determine how mutations in the Dbf4-RSIEGA sequence affected PBD binding. Dbf4-R83E, -I85A and -GA87AV peptides (containing mutations that disrupted the Dbf4-PBD two-hybrid interaction) lost the ability to compete with the Dbf4-PBD interaction even when the peptide concentration was increased to 10 !M (Figure 5D, E). However, the Dbf4-S84A and -E86K peptides, which still interacted with Polo in the two-hybrid assay, competed the Dbf4-PBD interaction in vitro (Figure 5D, E). These data are in complete agreement with the interaction map produced by two-hybrid data. Interestingly, the Dbf4-E86K peptide bound to the PBD with higher affinity than the wildtype peptide (10-100 nM, Figure 5E) and importantly, the Ser84 phosphorylated peptide lost the binding ability to PBD (Figure 5D). Therefore, although the S84A and S84E (phospho-mimetic) mutants did not noticeably affect the Dbf4-PBD interaction in the 49 two-hybrid assay (Figure 1C), a pS84 residue blocked the interaction in vitro. These data indicate that S84 phosphorylation is not simply dispensable for the Dbf4-PBD interaction, it blocks the interaction in vitro, suggesting that an entirely different type of PBD-protein interaction is occurring. Mutants altering critical residues in the Dbf4 PBD-binding motif suppress the cdc5-1 temperature sensitivity Deletion of the Dbf4 N-terminal 109 amino acids suppresses the temperature-sensitive (ts) lethal phenotype of the cdc5-1 mutant ((Miller et al. 2009), Figure 6A). Cdc5-1 protein contains a P511L missense mutation immediately preceding the PB1 motif and retains significant kinase activity at the non-permissive temperature (Pintard and Peter 2001) but is unable to promote mitotic exit (Park et al. 2003). The suppression data suggests that Dbf4 (DDK) might inhibit Cdc5-1 binding to critical targets required for mitotic exit. Using integrated alleles, we found dbf4-!82-88 and dbf4-R83E that are defective for the Polo interaction suppressed the cdc5-1 ts. The cdc5-1 mutant grew poorly at 30ºC, but the double mutants grew well at 30oC and also suppressed the cdc51 ts at 32ºC (Figure 6A). Although the dbf4-!82-88 and dbf4-R83E mutants suppressed the cdc5-1 ts at 32oC, there was little suppression at 34oC compared to the dbf4-N!109 mutant. This suggests that additional residues in the Dbf4 N-terminal 109 contribute to robust suppression of the cdc5-1 ts at 34oC. 50 Figure 6. Dbf4-RSIEGA mutants suppress the cdc5-1 temperature sensitivity (A) The indicated strains W303-1A, dbf4-!82-88 (M2805), cdc5-1 (M1614), cdc5-1 dbf4-!82-88 (M3112, and M3114), cdc5-1 dbf4-R83E (M3116, M3117), and cdc5-1 dbf4-N!109 (M2655, M2656) were spotted onto YPD plates and scored for growth at the indicated temperatures. (B) Various dbf4 deletions on an ARS CEN plasmid (pMW489) were introduced into M2600 (cdc5-1 dbf4!::kanMX6) and scored for growth by spotting serial dilutions on YPD media at the indicated temperatures. (C) Summary of dbf4 mutations, their effect on the Dbf4-PBD interaction, and suppression of the cdc5-1 ts. dbf4 mutants were scored for growth in the M2600 (cdc5-1 dbf4!::kanMX6) background by spotting serial dilutions on YPD media at increasing temperatures (Figure S4). (D) High copy plasmids expressing wild type DBF4 and the indicated mutants were transformed into M1614 (cdc5-1). Cultures were spotted onto SCM-Leu plates at 25oC indicating that high copy dbf4-N!65 lethality is alleviated by deleting residues 82-88. (E) Expression of the Dbf4 N-terminus from the GAL1, 10 promoter is lethal to cdc5-1 cells only if Dbf4 retains the ability to interact with Cdc5 as occurs in the WT, S84A, S84E, E86A and E86K mutants. 51 Figure 6. (cont’d) 52 Figure 6. (cont’d) 53 Figure 6. (cont’d) 54 Figure 6. (cont’d) 55 Figure 7. Mutations of Dbf4 residues required for the PBD interaction also suppress the cdc5-1 ts (A, B) A series of dbf4 mutants were shuffled into M2600 (dbf4!::kanMX6 cdc5-1) on plasmids and then cured of the wild type DBF4 plasmid. Serial dilutions of the resulting cultures were spotted at increasing temperatures to score the growth phenotype. Only those mutations that disrupt the PBD interaction suppress the cdc5-1 temperature sensitivity. Mutations such as SK92AA or S84A that have a wild type Dbf4-PBD interaction retain the cdc5-1 ts. The dbf4-E86K mutant actually causes enhanced cdc5-1 temperature sensitivity at 30oC, consistent with the increased interaction between the Dbf4 E86K peptide and the PBD (Figure 2E). 56 Figure 7. (cont’d) 57 To more closely examine the correlation between loss of the Polo interaction and the ability to suppress the cdc5-1 ts, we created a DBF4 plasmid shuffle system in the cdc51 background, and tested the ability of various dbf4 mutants (expressed from the endogenous DBF4 promoter) to suppress the cdc5-1 ts. We found that the dbf4-!66109, dbf4-!76-109, and dbf4-!82-109 alleles suppressed the cdc5-1 ts similar to the dbf4-N!109 allele. However, dbf4-!94-109 or dbf4-!100-109 that retain residues 83-88 did not (Figure 6B). These latter two mutants retain the ability to bind Cdc5 in the twohybrid assay (data not shown). In the cdc5-1 plasmid shuffle strain, the dbf4-N!109, dbf4-!66-109, dbf4-!76-109, and dbf4-!82-109 mutants exhibit very similar growth properties at 32oC (Figure 6B) but did not grow at 34oC (not shown). So in this system, the larger N-terminal deletion is phenocopied by smaller deletions removing the Cdc5 binding site. We then examined dbf4-R83A, -R83E, -I85A, -G87A, and -A88V alleles, which alter residues critical for PBD binding in the two-hybrid and AlphaScreen assays. As expected, these mutants suppressed the cdc5-1 ts at 30oC and 32ºC like the dbf4!82-88 cdc5-1 mutant (summarized in Figure 6C). In contrast, we observed no ts suppression by the dbf4-S84A and dbf4-E86K alleles, which still interacted with the PBD. We observed a strict correlation among mutants in residues 82-88 between loss of Polo binding and suppression of the cdc5-1 ts (Figure 6C). This indicates that loss of the Dbf4 physical interaction with Cdc5 suppresses the cdc5-1 growth defect at restrictive temperatures. The dbf4-S84A and dbf4-S84E mutants that still interact with Cdc5 did not suppress the cdc5-1 ts. Accordingly, the dbf4-E86K mutant exacerbated the cdc5-1 growth defect 58 (Figure 7B), consistent with the fact that the Dbf4-E86K peptide bound with higher affinity to the PBD than the wild type. These observations underscore that although alterations of Dbf4 residues S84 and E86 within the PBD-binding motif can influence the PBD interaction, the S84 and E86 residues do not make essential contacts required for the PBD interaction. Dbf4 inhibits Cdc5 by directly binding the PBD We previously found that a chromosomal dbf4-N!65 mutant that interacts with Cdc5 but is stabilized by deleting several D-boxes was synthetic sick or lethal in combination with cdc5-1 (Miller et al. 2009). This supports the model that Dbf4 inhibits the essential function of Cdc5. This hypothesis is supported by a recent report that a dbf4-N!65 mutant can inhibit ribosomal DNA segregation under certain circumstances (Sullivan et al. 2008), since rDNA segregation during anaphase is regulated by Cdc5 activation of the FEAR (Cdc14 Early Anaphase Release) pathway (Stegmeier and Amon 2004). We investigated whether Dbf4 residues 82-88 are required to inhibit Cdc5 activity by overexpressing Dbf4 from high-copy plasmids. Increased expression of wild-type DBF4 was deleterious to cdc5-1, but over-expression of dbf4-N!65 was lethal to cdc5-1 (Figure 6D). Deletion of residues 82-88 rescued the synthetic lethality between dbf4-N!65 and cdc5-1, strongly suggesting that Dbf4 inhibits Cdc5 function through a direct interaction. Similarly, overproduction of the isolated Dbf4 N-terminus (residues 1-225) from the GAL1, 10 promoter was lethal to cdc5-1 cells (Figure 6E) (Miller et al. 2009). In contrast, overproduction of the Dbf4-!82-88 and Dbf4-R83E peptides that fail to interact with Cdc5 was not lethal. Taken together with the cdc5-1 ts suppression results, these data 59 indicate that the Dbf4-RSIEGA sequence is required to inhibit Cdc5 activity by direct binding to the PBD. The PBD interacts with Dbf4 using a surface distinct from its phospho-peptide binding surface Among the Cdc5 PBD substrates that have been described in detail, the spindle pole body protein Spc72, was found to bind the PBD through its S-pS-P motif (Snead et al, 2007). We confirmed that purified His6-PBD bound the Spc72 phosphopeptide in vitro (Figure 8A) with an IC50 of ~2mM (data not shown) that is very similar to the Dbf4-PBD interaction. To test whether Dbf4 and Spc72 peptides bound to distinct surfaces of the PBD, we performed competition assays using a non-biotinylated Spc72 phosphopeptide to compete the Dbf4-PBD interaction. Although a wild type Dbf4 peptide spanning residues 80-93 effectively competed the Dbf4-PBD interaction, the phosphorylated Spc72 peptide (containing “S-pS-P”) did not (Figure 8B). This non-competitive result strongly suggests that two specific binding sites exist on the PBD, one for Dbf4 and another for phosphorylated substrates. 60 Figure 8. Dbf4 binds a surface on the PBD distinct from its phospho-protein binding site (A) A biotinylated Spc72 phospho-peptide (residues 223-242) bound the PBD in the AlphaScreen assay. (B) The same (non-biotinylated) Spc72 phospho-peptide did not compete the Dbf4-PBD interaction. (C) Purified wild type PBD and PBDHK proteins interact with Dbf4 in the AlphaScreen assay, but the PBD-HK mutant protein fails to interact with Spc72 phospho-peptide. (D) Two-hybrid Spc72(1-400) and Dbf4(66-227) interactions with the PBD were tested on the indicated plates. As in (C), mutation of the PBD “pincer” residues (H641A, K643M) or (W517F, H641A, K643M) had no affect on the Dbf4-PBD interaction, but eliminated the Spc72-PBD interaction. 61 Figure 8. (cont’d) AlphaScreen signal (1000x cps) A 50 Spc72-PBD interaction 40 30 20 10 0 6His-PBD 50 Biotin-Spc72 - 50 25 50 50 50 nM 50 100 200 nM B % Maximal counts Dbf4-PBD interaction 100 50 Dbf4-WT Spc72 (S-pS-P) 0 1 10 100 1000 10000 100000 competitor Non-biotinylated peptide (nM) 62 Figure 8. (cont’d) AlphaScreen signal (1000x cps ) C 60 40 20 0 biotin-peptide D PBD-WT PBD-HK Dbf4 Bait _ Dbf4 Spc72 _ (S-pS-P) Total cells Dbf4 Spc72 (S-pS-P) Y2H interaction Cdc5 Prey Spc72 Vector Vector PBD Spc72 PBD Spc72 PBD-HK Spc72 PBD-WHK Scm/-Trp-Leu Dbf4 Bait Scm/-Trp-Leu-His Total cells Y2H interaction Dbf4 Cdc5 Prey Vector Vector PBD Dbf4 PBD Dbf4 PBD-HK Dbf4 PBD-WHK Scm/-Trp-Leu Scm/-Trp-Leu-His +2mM 3AT 63 Figure 9. Identification of additional Cdc5 PBD mutations that disrupt the PBD-Spc72 interaction The Cdc5 PBD in pGAD-Cdc5.3 was randomly mutagenized using Taq polymerase. (A, B) PBDs were screened for their two-hybrid interaction with Dbf4 and Spc72 and this identified six discrete mutations that disrupt (Y618H, F526L, K643N, R620S) or impair (T515A, V537A) the interaction with Spc72 but have no effect on the PBD-Dbf4 interaction. (C) Description of mutations in Cdc5 and the corresponding amino acids in human Plk1. In the Plk1-phosphopeptide structures, L540 and R516 directly contact the phospho-threonine residue and peptide side chains, respectively (ref 24, 25) and S412 makes a water-mediated hydrogen bond with the phospho-threonine (ref 25). L423, V434, and W514 do not directly contact the peptide but are closely positioned to each other in space to make a hydrophobic region. Mutation of these residues may indirectly affect the phospho-peptide binding pocket. 64 Figure 9. (cont’d) 65 The co-crystal structure of the Plk1 PBD with a phosphothreonine peptide revealed that the “pincer” residues H538 and K540 (which are invariant among human and mouse Plk1, Polo, Plo1 and Cdc5) directly interact with the phosphate group on threonine (Cheng et al. 2003; Elia et al. 2003b). However, the purified PBD-H641A, K643M protein, containing the analogous mutations to Plk1-H538A, K540M, interacted with Dbf4 like the wild type in vitro (Figure 8C). In contrast, although the wild-type PBD interacted with Spc72 the PBD-HK protein did not. Similarly, the PBD-HK mutant interacted with Dbf4 but not with Spc72 in yeast cells (Figure 8D). The additional mutation of a conserved hydrophobic residue W517F, analogous to W414 in Plk1 that interacts with the preceding serine (S*-pT-P) of the phosphopeptide, also did not disrupt the two-hybrid interaction with Dbf4 (Figure 8D). Using random mutagenesis we isolated additional PBD mutations that abrogate the PBD-Spc72 interaction but retain the PBDDbf4 interaction (Figure 9). The effects of these new PBD mutants on phosphosubstrate binding are consistent with structural studies of Plk1-phosphopeptide molecular interactions (Cheng et al. 2003; Elia et al. 2003b). Together, these data indicate that the Cdc5 PBD contains a second binding surface that recognizes nonphosphorylated sequences, like the Dbf4 residues 83-RSIEGA-88. cdc5-HK “pincer” mutant has normal growth rate but shows increased resistance to microtubule disruption Very surprisingly, mutation of the pincer residues (H641A and K643M), which eliminated interaction with the Spc72 phospho-peptide in vitro (Figure 8C), was tolerated in yeast. In fact the cdc5-HK allele completely complemented yeast viability and growth in a 66 cdc5! plasmid shuffle strain (Figure 10A, B), and when integrated at the endogenous CDC5 locus (Figure 10C). Similarly, the cdc5-W517F, H641A, K643M (cdc5-WHK) allele also fully complemented yeast viability and growth rate (Figure 10A, B) and exhibited no temperature sensitivity up to 37oC (Figure 10A). These data indicate that the invariant pincer residues are not required for Cdc5 to bind essential substrates in yeast. Although there were little growth phenotypes, the cdc5-HK and cdc5-WHK mutants exhibited increased resistance over the wild type to microtubule disruption by benomyl (Figure 10C). All three strains grew well on plates containing 15 mg/ml benomyl but the mutant strains grew about 100-fold better in the presence of 30 and 37.5 mg/ml benomyl. This phenotype was also observed with the integrated cdc5-HK allele but not with the temperature sensitive (hypomorphic) cdc5-5 or cdc15-4 mutants, which were more sensitive or as sensitive to benomyl compared to the wild type strain (Figure 10C). These data raise the possibility that the PBD pincer residues target Cdc5 to a substrate (perhaps a microtubule associated protein) that regulates spindle dynamics. In M-phase, Cdc5 promotes loss of sister chromatid cohesion, regulates spindle dynamics, and is essential to promote mitotic exit (Archambault and Glover 2009). Therefore, we tested for cdc5-HK synthetic growth interactions with spindle checkpoint mutants and the cdc15, dbf2, and cdc14 ts MEN mutants. The cdc5-HK allele exhibited no synthetic growth interaction with the mad1D, mad2D, bub1D, or bub2D spindle checkpoint mutants (data not shown). Although we saw no synthetic growth interaction 67 with cdc14-1, cdc5-HK was synthetically lethal with cdc15-4, (cdc15-2, not shown) and dbf2-1 alleles (Figure 10D). These synthetic lethal interactions were alleviated by TAB61, a dominant cdc14 mutant that suppresses some MEN defects (Shou and Deshaies 2002). Importantly, TAB6-1 did not significantly affect the growth of the cdc15-2 or cdc15-4 mutants at 25oC (data not shown) arguing that TAB6-1 bypasses the synthetic lethality we observe by suppressing a cdc5-HK defect. This data therefore suggests that cdc5-HK is defective in promoting mitotic exit. Since cdc5-HK exhibited increased resistance to benomyl, we also directly examined spindle length in asynchronous wild type and cdc5-HK cells. The cell cycle distribution of cdc5-HK cells revealed a larger percentage of cells in G2/M phase relative to the wild type suggesting a mitotic delay in the mutant (Figure 11A). We quantitated spindle length in large-budded, mitotic cells and observed that the fraction of cells with short spindles (<2mm) was the same in both strains, indicating there was no defect in mitotic entry (Figure 11B). In contrast, the average spindle length in the mutant was about 38% greater than the wild type (7mm versus 5.1mm) (Figure 11B). This could indicate a defect in exiting mitosis (spindle disassembly) or a defect in properly restraining spindle elongation. To address whether spindle elongation occurred with faster kinetics in the mutant, we measured the rate of spindle growth following release from a G2/M block using nocodazole. Wild type and cdc5-HK cells were arrested for three hours using 15 mg/ml nocodazole and then released into the cell cycle in the absence of nocodazole at 30oC. Flow cytometry profiles are shown in Figure 11C. The spindles in both strains were depolymerized at 0 minutes, however spindle length increased more rapidly for the 68 cdc5-HK mutant varying from 300% over wild type lengths at early time points to 40% greater length at 60 minutes (Figure 11D, E). These data indicate that mutation of the pincer residues also causes aberrant spindle elongation. 69 Figure 10. The Cdc5 pincer residues are not required for yeast viability (A) The cdc5-HK and cdc5-WHK mutants complemented a cdc5! by plasmid shuffle into M1672 (cdc5!::kanMX6/pMW536[CDC5 URA3]) evidenced by growth on FOA plates, and at various temperatures on YPD plates following loss of pMW536. (B) Growth curves of M1672 strains containing only the indicated CDC5 alleles in YPD at 30oC (C) The M1672-transformed strains in panel (A) were spotted onto YPD plates +/- benomyl (top). WT (M138), cdc5-HK (M3502), cdc5-5 (M1680), and cdc15-4 (M1999) strains containing integrated alleles were similarly spotted onto YPD +/benomyl (bottom). (D) Representative tetrads from diploid strains of genotype cdc5-HK/CDC5 dbf2-1/DBF2, cdc5HK/CDC5 cdc15-4/CDC15 TAB6-1/CDC14, or cdc5-HK/CDC5 cdc14-1/CDC14 that were sporulated and dissected onto YPD plates at 25oC. Recombinant genotypes are indicated. 70 Figure 10. (cont’d) 71 Figure 10. (cont’d) 72 Figure 10. (cont’d) 73 Figure 11. Mutation of the Cdc5 pincer residues causes a G2/M delay and alters spindle dynamics (A) Flow cytometry profiles of asynchronous W303-1A (WT) and M3502 (cdc5-HK) strains. (B) Average spindle length was quantitated in large-budded cells of the same strains, shown with the range that includes 25-75% of spindle lengths. Inset shows fraction of cells with short spindles, <2 mm. (C) Flow cytometry profiles of W303-1A and M3502 arrested in G2 with nocodazole for 3 hours (t=0) and following release at 30oC. (D) Quantitation of spindle length at the indicated times following nocodazole release. Standard errors were all less than 1%. (E) Tubulin staining of representative photomicrographs of cells at 40 minutes following nocodazole release. 74 Figure 11. (cont’d) 75 Figure 11. (cont’d) 76 Figure 11. (cont’d) 77 Figure 12. The dbf4-!82-88 mutant exhibits normal cell cycle progression. Wild-type W303- 1A (M138) and dbf4-!82-88 (M2804) strains were arrested in G1 phase with alpha factor, and then released into cell cycle at 25oC. Samples were collected at indicated time points and analyzed by flow cytometry. 78 DISCUSSION In this study, we determined the Dbf4 residues required for a physical interaction with Cdc5. This analysis revealed a novel Dbf4 sequence (83-RSIEGA-88) directly binds the PBD. Unlike most identified PBD-binding sites, the Dbf4-PBD interaction did not require Ser/Thr phosphorylation and notably, bound to a distinct binding surface within the PBD. Our results establish that Dbf4 residues 83-88 are critical for binding the polo-box domain and mediate an inhibition of Cdc5 function. Surprisingly, the ability of Cdc5 (PBD) to interact with phosphorylated substrates is apparently not required for normal yeast growth but is required for full MEN activation and for regulating spindle dynamics during mitosis. An alternative mode of PBD binding The polo-box domains of human Plk1 and yeast Cdc5 bind proteins containing phospho-serine or phospho-threonine consensus sites. In the co-crystal structure of the Plk1-PBD with a phosphorylated peptide, the peptide binds to a shallow pocket at the interface between two polo-box motifs, called PB1 and PB2 (Cheng et al. 2003; Elia et al. 2003b). Two highly conserved residues, H538 and K540 in PB2, directly contact the phosphate group. Further mutational and biological studies have confirmed that the PBD binds phosphorylated substrates in vivo and that the PBD is required for Plk1 function in human cells and in yeast, where it complements CDC5 activity (Lee and Erikson 1997; Ouyang et al. 1997). 79 The optimal phosphopeptide binding motif containing Ser-[pSer/pThr]-[Pro/X] was described for Cdc5 substrates but only a few of these binding sequences have been characterized or mapped in detail (Yoshida et al. 2006; Lowery et al. 2007; Crasta et al. 2008). Although a subset of Cdc5 substrates examined in one study were found to be phosphorylated by Cdks (Lee et al. 2005), additional Cdc5 substrates might be primed by other kinases or by Cdc5 itself, as has been recently shown for Plk1 (Lee et al. 2008). In contrast, the PBD of Drosophila Polo was recently shown to mediate an interaction with the microtubule-associated protein Map205 without a requirement for Map205 phosphorylation (Archambault et al. 2008). In addition, the Cdc5 PBD can bind to Cdc14 independently of a consensus PBD recognition site (Rahal and Amon 2008). We defined a unique Dbf4 sequence (83-RSIEGA-88) that directly interacts with the PBD. This Dbf4 sequence differs from the PBD consensus-binding sequence in two critical regards. Firstly, this sequence does not contain the absolutely conserved serine preceding a potential phospho-serine or -threonine residue. More importantly, serine phosphorylation is not required for the PBD interaction since mutation of S84 to alanine had little effect on PBD binding in vitro or in vivo. In fact, a peptide containing phosphorylated S84 lost the ability to compete the Dbf4-PBD interaction in vitro (Figure 5D). Therefore, S84 phosphorylation actually inhibited interaction with the PBD. If S84 phosphorylation occurs in vivo, this might negatively regulate the DDK-Cdc5 interaction. Whether a similar “RSIEGA” sequence exists in Cdc5 substrates remains to be determined, but our data strongly suggests that the PBD utilizes a second mode of interaction to bind non-phosphorylated proteins. The PBD-HK mutant protein bound 80 Dbf4 as the wild type, but was defective for interaction with the consensus (S-pS-P) Spc72 peptide. Mutation of six additional PBD residues (three of which mediate Plk1::phospho-peptide contacts in the co-crystal structure) disrupted the PBD-Spc72 canonical interaction but had no effect on the PBD-Dbf4 interaction (Figure 9). Furthermore, the Dbf4-PBD interaction was not competed by a phosphorylated consensus peptide (Figure 8B). These data indicate that the Cdc5 PBD can interact with proteins using two different binding surfaces, one that recognizes phosphorylated substrates and one that recognizes Dbf4. Although the Plk1-HK mutant is defective for phospho-peptide binding in vitro and Plk1 activity in vivo, we found that the analogous cdc5-H641A, K643M mutant in budding yeast had a wild type growth rate and exhibited no temperature sensitivity (Figure 10A, B). This mutant instead exhibited a G2/M delay by flow cytometry, increased resistance to benomyl, and had an elongated spindle phenotype. These data suggest that Cdc5HK protein is defective for interactions that restrain spindle elongation or that promote spindle disassembly. Since spindle disassembly follows Cdc5 activation of the MEN (Stegmeier and Amon 2004), a defect in MEN activation could account for the longer spindle length in the mutant. The synthetic lethality of cdc5-HK with cdc15 or dbf2 ts mutants supported the idea that cdc5-HK has a defect in MEN activation. However, arguing against the MEN defect per se causing increased resistance to benomyl, we found that the cdc5-5 and cdc15-4 MEN mutants had similar or greater sensitivity to benomyl than wild type. The cdc5-1 and cdc5-2 mutants also exhibited a greater sensitivity to benomyl than wild type (data not shown). The wild type growth rate and 81 unique resistance to benomyl argue that the HK mutation caused another defect in Cdc5 activity and that cdc5-HK was not simply another hypomorphic MEN mutant. The increased rate of spindle elongation in the mutant compared to the wild type following release of arrested G2/M cells strongly suggests that the cdc5-HK mutant has altered spindle dynamics. These data indicate that the pincer residues are not required for the essential function of Cdc5 and since the PBD-HK mutant does not bind phosphopeptides in vitro, strongly suggest that PBD interactions with phospho-proteins are not essential in yeast. Dbf4 is a scaffold for Cdc5 inhibition When a Cdc5-Dbf4 two-hybrid interaction was first described it was proposed that Cdc5 might have a novel role in DNA replication, and that Dbf4 possibly functioned as a scaffold between these two essential kinases (Hardy and Pautz 1996). Although Cdc5 is absent in G1 and early S-phase phase (Charles et al. 1998; Cheng et al. 1998; Shirayama et al. 1998), it could potentially influence DNA replication during the preceding mitosis. However when cells are released from a G1 block in the absence of Cdc5 expression, DNA synthesis occurs on schedule and cells arrest in telophase with segregated chromatids (Hu et al. 2001; Yoshida et al. 2006; Liang and Wang 2007). The Xenopus Plk1 ortholog, Plx1, was recently shown to influence DNA replication in response to DNA damage raising the possibility that additional Polo orthologs might have a similar role (Trenz et al. 2008). We recently proposed that DDK inhibits Cdc5 function during mitotic exit through a direct Dbf4-Cdc5 interaction (Miller et al. 2009). Here we show that multiple dbf4 mutants defective the Dbf4-Cdc5 interaction suppress 82 the cdc5-1 temperature sensitivity. Furthermore, increased Dbf4 expression is lethal to cdc5-1 cells at the permissive temperature but only if Dbf4 can bind to Cdc5 (Figure 11D). These data indicate that Dbf4 inhibits Cdc5 function by direct association with the PBD. Since DDK phosphorylates Cdc5 in vitro (Miller et al. 2009), these findings suggest that Dbf4 may serve as scaffold in late S-phase so that Cdc7 kinase can inhibit Cdc5 by phosphorylating Cdc5 or an essential Cdc5 substrate. It is also possible that Dbf4 inhibits Cdc5 simply by binding to the PBD to prevent access to essential mitotic substrates, since overproduction of a Dbf4 N-terminal peptide is lethal to cdc5-1 (Figure 11E, and ref (Miller et al. 2009)). The DDK regulation of Cdc5 is not required for cell division control under normal conditions, since cell cycle progression occurs normally in dbf4 mutants defective for the Cdc5 interaction in otherwise wild-type cells (Figure 12 and ref (Gabrielse et al. 2006)). This suggests redundant mechanisms to delay Cdc5 activation until anaphase onset. For instance, Kin4 kinase antagonizes Cdc5 function when mitotic spindle positioning errors occur, although this may occur through inhibitory phosphorylation of Cdc5 substrates and not direct Cdc5 phosphorylation (D'Aquino et al. 2005; Pereira and Schiebel 2005). DNA damage and the Rad53 checkpoint kinase also block mitotic exit by directly or indirectly affecting Cdc5 activity (Sanchez et al. 1999; Liang and Wang 2007). Interestingly, the dbf4-N!109 deletion mutant that lacks the Polo binding site is synthetically lethal with rad53-1 (Gabrielse et al. 2006) raising the possibility that Dbf4 and Rad53 have redundant, but together essential, roles to inhibit Cdc5 during an unperturbed cell cycle. 83 In summary, we have uncovered a novel PBD binding motif in Dbf4 that may be conserved in other PBD binding proteins. We suggest that DDK uses this unique motif to bind Cdc5 and inhibit its essential function in the mitotic cell cycle. Presumably the Dbf4-PBD interaction does not preclude binding to substrates containing a phosphorylated consensus site based on our peptide competition studies. Therefore, the DDK-Cdc5 ternary complex could in principal interact with Cdc5 substrates containing a phospho-PBD consensus sequence. This model agrees with the finding that DDK and Cdc5 interact during meiosis and that both proteins phosphorylate the Cdc5 substrate Mam1 to promote monopolar spindle orientation during meiosis I (Matos et al. 2008). Defining the Dbf4-PBD physical interaction allows a rigorous investigation of how DDK regulates mitotic and meiotic events. 84 MATERIALS AND METHODS Construction of Yeast Strains, Plasmids and Baculoviruses Strains and plasmids used in this study are listed in Tables 1 and 2. PJ69-4a cells (MATa trp1-901 leu2-3,112 ura3-52 his3-200 gal4! gal80! LYS2::GAL1-HIS3 GAL2ADE2 met::GAL7-lacZ) were used for two-hybrid experiments. All other strains were derivatives of W303 (MATa ade2-1 trp1-1 can1-100 leu2-3, 112 his3-11, 15 ura3). The CDC5 shuffle stain M1672 (cdc5!::kanMX6/pMW536 [CDC5 URA3 ARS-CEN]) was constructed using the same procedure for the DBF4 shuffle strain, M895, as previously described (Gabrielse et al. 2006). To integrate dbf4 mutants, HindIII-XbaI fragments containing full length dbf4-!82-88 or dbf4-R83E were co-transformed into M895 (dbf4!::kanMX6/pMW490 [DBF4 URA3 ARS-CEN]) together with pRS415. Leu-positive transformants were replica plated on FOA. Multiple FOA resistant colonies were recovered to YPD plates and then tested on YPD plates containing 0.2 mg/ml Geneticin to score loss of the kanMX6 marker. The resulting Geneticin-sensitive candidates were confirmed as correct recombinants following PCR amplification of the DBF4 locus and then backcrossed to W303. The epitope-tagged Cdc5 strains were made by the method of Longtine (Longtine et al. 1998). Deletions and point mutations within DBF4 and CDC5 were generated by site-directed mutagenesis using the QuikChange system (Stratagene). PCR amplified NcoI-PstI fragments containing the full-length DBF4 coding sequence or various dbf4 mutants were cloned into the same sites of pGBKT7 (Clonetech) to give the GalDBD-Dbf4 fusions. CDC5 (-332 to +2360) was PCR amplified from genomic DNA and cloned into the 85 HindIII-XbaI sites of pRS415 and pRS416 to give pMW535 and pMW536, respectively. Spc72 residues 1-400 were PCR amplified from genomic DNA and cloned into the NdeI-BamHI sites of pGBKT7 to give pYJ356. For high-copy number plasmids, HindIIINotI fragments containing entire WT DBF4 or various dbf4 mutants were cloned into the same sites of pRS425. Cdc5 residues 357-705 were cloned on a BamHI-XhoI into pET24a-GST (gift of Eric Xu, Van Andel Research Institute) for expression of His6-GSTPBD. Construction of baculovirus plasmids encoding WT Dbf4, Dbf4-N!109, HA-Cdc7, and 3Myc-Cdc5 was previously described (Gabrielse et al. 2006). An NcoI-NotI fragment containing dbf4-!82-88 was cloned in the baculovirus transfer vector, pAcSG2. Hightiter baculoviruses were generated by transfection of Sf9 cells using the BaculoGold kit (BD Biosciences) followed by plaque purification and virus amplification. Growth Conditions, Cell Cycle Synchronization, and Immunofluorescence Cells were cultured in YPD (1% yeast extract, 2% bacto peptone and 2% glucose). Synthetic Complete Medium (SCM) (Sherman et al. 1986) was supplemented with 5% glucose or 2.5% galactose. Benomyl (Sigma) was added directly to plates immediately before pouring (final 0.2% DMSO (v/v)). Synchronous G1 or G2/M cultures were obtained after addition of 5µg/ml of alpha-factor or 15mg/ml nocodazole, respectively, for 3 hours. DNA content was analyzed by flow cytometry as previously described (Weinreich and Stillman 1999). Tubulin and DAPI staining was previously described (Soues and Adams 86 1998). Spindle length was measured by 60x objective using a Nikon Eclipse TE300 fluorescence microscope and OpenLab version 3.1.7 image analysis software. Two-hybrid Analysis Various DBF4 bait constructs containing Gal4 DNA binding domain (DB) were transformed with pGAD-Cdc5.3 (Gal activation domain (AD) fusion to Cdc5357-705) in PJ69-4a and selected on SCM plates lacking tryptophan and leucine. These were spotted at ten-fold serial dilutions on the same plates and also on plates also lacking histidine but containing 2 mM 3-aminotriazole (3AT) at 30°C and cultured for 2-3 days. Yeast Whole-cell Extracts, IP from Sf9 cells, and Western Blotting Yeast protein extracts were prepared for Western blotting by trichloroacetic acid extraction (Foiani et al. 1994). Blots were probed in phosphate-buffered saline containing 0.1% Tween containing 1% dried milk. Dbf4 bait constructs contained a Myc tag were detected using anti-Myc monoclonal antibody (9E10, 1:2000) followed by antimouse-HRP secondary antibody. Sf9 cells were co-infected with HA-Cdc7, 3Myc-Cdc5 and Dbf4 mutants as previously described (Gabrielse et al. 2006). Whole cell extracts and IPs were probed with polyclonal antibodies against Cdc7 (1:4000) and Dbf4 (1:1000). 3Myc-Cdc5 was detected with 9E10. Protein Purification and Peptide Binding Assays His6-GST-Cdc5 (PBD) was induced in BL21 cells for 3 hours at 30°C using 0.5 mM IPTG. Cells were sonicated in PBS containing 1% Triton X-100 and GST proteins were 87 purified from soluble extracts by binding to glutathione-agarose (Amersham) and eluted in the buffer (20 mM Tris-HCL, 150 mM NaCl, 1 mM EDTA and 10% glycerol) containing 5mM glutathione and dialyzed against 50 mM MOPS (pH 7.4), 100 mM NaCl, and 10% glycerol. Dbf4 peptide-PBD binding was quantitated using the AlphaScreen luminescence proximity assay (PerkinElmer Life Sciences) using a histidine detection kit as described (Li et al. 2008). Binding mixtures containing 50 nM N-terminally biotinylated Dbf4 peptide (Biotin-EKKRARIERARSIEGAVQVSKGTG), 50 nM 6His-GST-PBD, 15 µg/ml of streptavidin-coated donor beads, and Ni-chelate-coated acceptor beads, were incubated in buffer containing 50 mM MOPS (pH 7.4), 100 mM NaCl, 0.1 mg/ml BSA for 1 h. Luminescence was recorded in a 384-well plate using an Envision 2104 plate reader (PerkinElmer Life Sciences). For competition assays, titrated unlabeled peptides were added and incubated at room temperature for 1 hour before measurement. Nonlinear regression as implemented in Prism 5.0 (GraphPad Software, San Diego) was used to fit the data to a variable slope dose-response inhibition equation to determine IC50 values. All peptides used in this study are listed in Table 3. 88 Table 1. Plasmids Plasmid pAcSG2 pCG10 pCG40 pCG53 pCG60 pCG74 pCG162 pCG166 pCG213 pCM1 pCM16 Description pRS415-DBF4110-704 pAcSG2-DBF4110-704 pGBKT7-Dbf466-227 pCG53 ADH1 promoter-!(-732)-(-802) pGBKT7-Dbf4110-704 pRS416-pGAL1,10 pCG162-DBF41-225 pCG162-DBF41-225 !82-88 pGAD-C1-CDC5421-705 H641A K643M pAcSG2-3myc-CDC565-705 pET24a-GST pGAD-C1 pGADCdc5.3 pGBKT7 pJK17 pMW1 pMW47 pMW489 pMW490 pMW526 pMW535 pMW536 pMW541 pRS415 pRS416 pRS425 pYJ1 pYJ2 pYJ3 pYJ4 pYJ5 pYJ6 pYJ7 pYJ8 pYJ9 pYJ10 pGAD-C1-CDC5421-705 Source BD Biosciences Gabrielse et al., 2006 Miller et al., 2009 Miller et al., 2009 Miller et al., 2009 Miller et al., 2009 Miller et al., 2009 Miller et al., 2009 Miller et al., 2009 This study Miller et al., 2009 Eric Xu, Van Andel Institiute, MI James et al. 1996 Miller et al., 2009 Clontech This study Gabrielse et al., 2006 Gabrielse et al., 2006 Gabrielse et al., 2006 Gabrielse et al., 2006 Gabrielse et al., 2006 This study This study This study Sikorski and Hieter, 1989 Sikorski and Hieter, 1989 Sikorski and Hieter, 1989 This study This study This study This study This study This study This study This study This study This study pGAD-Cdc5.3 Y618H pAcPK30-DBF4 pAcSG2-HAHIS6-CDC7 pRS415-DBF41-704 pRS416-DBF41-704 pRS415-DBF466-704 pRS415-CDC51-705 pRS416-CDC51-705 pMW535 H641A K643M LEU2 ARS-CEN URA3 ARS-CEN LEU2 2"m pCG60-DBF472-227 pCG60-DBF477-227 pCG60-DBF482-227 pCG60-DBF488-227 pCG60-DBF494-227 pCG60-DBF4100-227 pCG60-DBF4104-227 pCG60-DBF4108-227 pCG60-DBF4110-227 pCG60 R83E S84A 89 Table 1. (cont’d) pYJ11 pYJ13 pYJ14 pYJ15 pYJ16 pYJ17 pYJ18 pYJ19 pYJ20 pYJ21 pYJ22 pYJ26 pYJ28 pYJ30 pYJ32 pYJ33 pYJ34 pYJ36 pYJ38 pYJ40 pYJ46 pYJ47 pYJ49 pYJ53 pYJ56 pYJ59 pYJ61 pYJ65 pYJ67 pYJ68 pYJ74 pYJ79 pYJ83 pYJ84 pYJ88 pYJ100 pYJ111 pYJ114 pYJ123 pYJ124 pYJ126 pCG60 I85A E86K pCG60 S92A K93E pCG60 V89A Q90A V91A pCG60 S84A pCG60 R83A S84A pCG60 I85A E86A pCG60 G87A A88V pMW489 V89A Q90A V91A pMW489 S92A K93E pMW489 I85A E86K pCG60-DBF466-96 pCG60 A82V pCG60 R83A pCG60 R83E pCG60 I85A pCG60 E86K pCG60 G87A pCG60 A88V pCG60-DBF466-227 !82-88 pCG60 E86A pMW489 R83E S84A pYJ22-DBF472-96 pYJ22-DBF488-96 pMW489 S84A pMW489 G87A A88V pYJ22-DBF477-96 pYJ22-DBF466-96 !82-88 pMW489 A82V pMW489 I85A pMW489 G87A pMW489-DBF4!82-88 pMW489 A88V pET24a-GST-CDC5357-705 pMW489 R83E pMW489 E86K pCG60-DBF466-227 !82-96 pMW489 R83A pMW489 E86A pCG60 S84E pMW489 S84E pYJ22-DBF466-96 78-81x4A 90 This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study Miller et al., 2009 This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study Table 1. (cont’d) pYJ128 pYJ136 pYJ137 pYJ139 pYJ141 pYJ143 pYJ145 pYJ148 pYJ150 pYJ152 pYJ153 pYJ154 pYJ157 pYJ160 pYJ165 pYJ167 pYJ169 pYJ171 pYJ174 pYJ182 pYJ189 pYJ193 pYJ195 pYJ198 pYJ201 pYJ204 pYJ206 pYJ210 pYJ211 pYJ212 pYJ215 pYJ218 pYJ221 pYJ222 pYJ231 pYJ236 pYJ237 pYJ238 pYJ260 pYJ263 pYJ272 pCG60-DBF466-227 78-81x4A pCG60 V89A pCG60 Q90A pCG60 G94A T95A G96A pMW489 V89A pMW489 G94A T95A G96A pMW489 Q90A pMW489 S92A pRS425-DBF4110-704 pRS425-DBF41-704 !82-88 pRS425-DBF41-704 G87A A88V pRS425-DBF41-704 pRS425-DBF466-704 pRS425-DBF41-704 R83E pCG60 S92A K93A pCG60 S92A pCG60 K93E pMW489 K93E pRS425-DBF41-704 E86K pAcSG2-DBF41-704 !82-88 pRS425-DBF466-704 !82-88 pMW489-DBF4!76-109 pMW489-DBF4!82-109 pMW489-DBF4!66-109 pMW489-DBF466-704 !82-88 pGBKT7-Dbf41-704 pGBKT7-Dbf41-704 !82-88 pGBKT7-Dbf41-704 !66-109 pGBKT7-Dbf41-704 !76-109 pGBKT7-Dbf41-704 !82-109 pGBKT7-Dbf466-704 !82-88 pMW489-DBF4!89-109 pMW489-DBF4!100-109 pMW489-DBF4!94-109 pMW489 S92A K93A pMW489-DBF4!89-93 pMW489-DBF4!89-91 pMW489-DBF4!91-93 pRS425-DBF41-704 S84A pRS425-DBF41-704 S84E pRS425-DBF41-704 V89A 91 This study This study This study This study This study This study This study This study Miller et al., 2009 Miller et al., 2009 This study Miller et al., 2009 This study This study This study This study This study This study This study This study This study This study This study This study This study Miller et al., 2009 Miller et al., 2009 This study This study This study Miller et al., 2009 This study This study This study This study This study This study This study This study This study This study Table 1. (cont’d) pYJ274 pYJ276 pYJ278 pYJ292 pYJ294 pYJ296 pYJ297 pYJ298 pYJ302 pYJ303 pYJ314 pYJ316 pYJ326 pYJ327 pYJ328 pYJ356 pYJ365 pYJ368 pYJ415 pYJ439 pYJ441 pYJ443 pYJ445 pYJ447 pRS425-DBF41-704 Q90A pRS425-DBF41-704 S92A pRS425-DBF41-704 K93E pCG166 R83E pCG166 S84A pCG166 S84E pCG166 E86A pCG166 E86K pCG60-DBF466-227 !82-93 pMW489-DBF4!82-93 pCM1 W517F pMW541 W517F pCG60-DBF466-227 !89-93 pCG60-DBF466-227 !89-91 pCG60-DBF466-227 !92-93 pGBKT7-Spc721-400 pYJ83 H641A K643M pYJ83 W517F H641A K643M pCG60 V91A pGAD-Cdc5.3 F526L pGAD-Cdc5.3 K643N pGAD-Cdc5.3 R620S pGAD-Cdc5.3 T515A pGAD-Cdc5.3 V537A 92 This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study Table 2. Yeast strains Stain W303-1A PJ69-4A M319 M331 M895 M1614 M1649 M1656 M1678 M1680 M1672 M1800 M1999 M2600 M2655 M2657 M2804 M2805 M2806 M2807 Genotype MATa ade2-1, ura3-1 his3-11, -15 trp1-1 leu2-3, -112 can1100 MATa trp1-901 leu2-3, -112 ura3-52 his3-200 gal4! gal80! LYS2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZ W303 MATa dbf2-1 W303 MATa cdc15-2 W303 MATa dbf4!::kanMX6 [pMW490; pRS416-DBF4 URA3] W303 MATa cdc5-1 W303 MATa cdc14-1 W303 MATa dbf4-N!109-kanMX6 W303 MATa cdc5-2(msd2-1)-URA3 W303 MATa cdc5-5(msd2-4)-URA3 W303 MATa cdc5!::kanMX6 [pMW536; pRS416-CDC5 URA3] W303 MAT! dbf4-N!109-kanMX6 W303 MATa cdc15-4 W303 MATa cdc5-1 dbf4!::kanMX6 [pMW490; pRS416DBF4 URA3] W303 MATa cdc5-1 dbf4-N!109-kanMX6 W303 MAT! cdc5-1 dbf4-N!109-kanMX6 W303 MATa dbf4-!82-88-kanMX6 W303 MAT! dbf4-!82-88-kanMX6 W303 MATa dbf4-R83E-kanMX6 W303 MAT! dbf4-R83E-kanMX6 93 Source Thomas and Rothstein, 1989 James et al., 1996 Miller et al., 2009 Miller et al., 2009 Cabrielse at al., 2006 Miller et al., 2009 Miller et al., 2009 Miller et al., 2009 This study This study This study Miller et al., 2009 This study This study Miller et al., 2009 Miller et al., 2009 Miller et al., 2009 This study This study This study Table 2. (cont’d) M3112 M3114 M3116 M3117 M3376 M3377 M3378 M3486 M3490 M3502 M3526 W303 MATa cdc5-1 dbf4-!82-88-kanMX6 W303 MAT! cdc5-1 dbf4-!82-88-kanMX6 W303 MATa cdc5-1 dbf4-R83E-kanMX6 W303 MAT! cdc5-1 dbf4-R83E-kanMX6 W303 MATa cdc5!::kanMX6 [pMW535; pRS415-CDC5 LEU2] W303 MATa cdc5!::kanMX6 [pMW541; pRS415-cdc5H641A-K643M LEU2] W303 MATa cdc5!::kanMX6 [pYJ314; pRS415-cdc5W517F-H641A-K643M LEU2] W303 MATa cdc5-H641A-K643M W303 MATa TAB6-1 W303 MATa cdc5-H641A-K643M-kanMX6 W303 MATa TAB6-1-TRP1 94 Miller et al., 2009 This study This study This study This study This study This study This study D’Aquino et al., 2005 This study This study Table 3. Peptides Peptide name Biotin-Dbf4 7396 Dbf4 73-96 Dbf4 78-96 Dbf4 78-93 Dbf4 80-93 Dbf4-R83E Dbf4-S84A Dbf4-pS84 Dbf4-I85A Dbf4-E86K Dbf4-GA87AV Biotin-Spc72 Spc72 Abbr. Biotin-p p1 p2 p3 p4 R83E S84A pS84 I85A E86K GA87AV BiotinSpc72 Spc72 Peptide sequence Biotin-EKK RAR IER ARS IEG AVQ VSK GTG EKK RAR IER ARS IEG AVQ VSK GTG RIE RAR SIE GAV QVS KGT G RIE RAR SIE GAV QVS K ERA RSI EGA VQV SK ERA ESI EGA VQV SK ERA RAI EGA VQV SK ERA R(pS)I EGA VQV SK ERA RSA EGA VQV SK ERA RSI KGA VQV SK ERA RSI EAV VQV SK Length MW Biotin + 24 24 19 16 14 14 14 14 14 14 14 2854 2627.9 2015.3 1799.2 1530 1503.1 1514.1 1610.1 1487.7 1529.2 1572.2 Biotin-EEF LSL AQS (pS)PA GSQ LES RD EEF LSL AQS (pS)PA GSQ LES RD Biotin + 20 20 2457.6 2231.3 95 ACKNOWLEDGMENTS We thank the Van Andel Research Institute and the American Cancer Society (RSG0506301GMC) for supporting this research; the Flow Cytometry lab for technical assistance; FuJung Chang and Carrie Gabrielse for technical help. 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The use of in vitro peptide-library screens in the analysis of phosphoserine/threonine-binding domain structure and function. Annu Rev Biophys Biomol Struct 33: 225-244. Yoshida, S., Kono, K., Lowery, D.M., Bartolini, S., Yaffe, M.B., Ohya, Y., and Pellman, D. 2006. Polo-like kinase Cdc5 controls the local activation of Rho1 to promote cytokinesis. Science 313(5783): 108-111. 103 CHAPTER 3 RAD53 BINDS DBF4 THROUGH AN N-TERMINAL T-X-X-E MOTIF AND THIS INTERACTION IS REQUIRED TO SUPPRESS LATE ORIGIN FIRING Ying-Chou Chen, Jessica Kenworthy, Christine Hänni, Philip Zegerman, and Michael Weinreich The work presented in this chapter has been submitted to Genetics. 104 RAD53 BINDS DBF4 THROUGH AN N-TERMINAL T-X-X-E MOTIF AND THIS INTERACTION IS REQUIRED TO SUPPRESS LATE ORIGIN FIRING ABSTRACT Cdc7-Dbf4 (DDK) and cyclin dependent kinase (CDK) are essential to initiate DNA replication at individual origins. During replication stress, the S-phase checkpoint inhibits the DDK- and CDK-dependent activation of late replication origins. The Rad53 kinase is a central effector of the replication checkpoint, and both binds to and phosphorylates Dbf4 to prevent late origin firing. The molecular basis for the Rad53Dbf4 physical interaction is not clear but occurs through the Dbf4 N-terminus. Here, we have characterized the molecular interaction between Dbf4 and Rad53. Surprisingly, both Rad53 FHA domains bind Dbf4 through the same N-terminal T105-x-x-E motif, which closely resembles an optimal pT-x-x-D FHA1 binding site. This sequence precedes a conserved BRCT domain in Dbf4, which is also required for the interaction with Rad53. The Rad53 FHA1 domain correspondingly binds pT-x-x-E (but not T-x-x-E) Dbf4 peptides in vitro. Abrogation of the Rad53-Dbf4 physical interaction allows late origin firing during replication checkpoint activation. One model to explain these data is that activated Rad53 uses both its FHA domains to bind two separate pT105-x-x-E sequences within a Dbf4 multimer. Rad53-Dbf4 docking then allows Rad53 to phosphorylate Dbf4 at critical C-terminal sites, which in turn block late origin firing during periods of genome stress. 105 INTRODUCTION The fidelity of chromosome replication depends on checkpoint mechanisms to stabilize stalled forks, regulate origin activation, and repair DNA damage (Hartwell and Weinert 1989; Bartek et al. 2004; Segurado and Tercero 2009). In response to replication stress, the replication checkpoint maintains replisome stability and prevents late origins from firing, which allows time for DNA repair and the completion of DNA replication prior to chromosome segregation. Incomplete DNA replication or uncoordinated origin firing following DNA damage can result in genomic instability, cancer predisposition, and premature aging (Branzei and Foiani 2010). In the budding yeast Saccharomyces cerevisiae, activation of the checkpoint sensor kinase, Mec1 (vertebrate ATR), is triggered at stalled forks or sites of DNA damage (Majka et al. 2006; Labib and De Piccoli 2011). Subsequent signal amplification through the Mrc1 or Rad9 adaptors leads to activation of the checkpoint kinase Rad53 (the ortholog of the human tumor suppressor Chk2) (Branzei and Foiani 2009). Rad53 is an integral transducer of various cellular responses to replication stress or DNA damage. Rad53 induces a series of transcriptional responses through MBF-regulated genes (Bastos de Oliveira et al. 2012; Travesa et al. 2012) and also activates the Dun1 kinase, which promotes the expression of ribonucleotide reductase (RNR) subunits and additional DNA repair genes (Huang et al. 1998). In parallel, Rad53 down-regulates the RNR inhibitor Sml1 to increase deoxyribonucleotide levels and facilitate DNA synthesis (Zhao et al. 2001). In response to replication fork stalling, Rad53 prevents the activation of late replication origins by phosphorylating two proteins required for the initiation of 106 DNA replication: Dbf4 and Sld3 (Lopez-Mosqueda et al. 2010; Zegerman and Diffley 2010; Duch et al. 2011). Dbf4 is the regulatory subunit of Cdc7 kinase, which is required for initiating DNA replication at individual origins by phosphorylating the replicative MCM helicase (Tsuji et al. 2006; Francis et al. 2009; Randell et al. 2010; Sheu and Stillman 2010). Sld3 is also required for activating the MCM helicase by promoting Cdc45-MCM association (Fu and Walter 2010; Boos et al. 2011). Cdc7 requires the Dbf4 regulatory subunit for kinase activity. Dbf4 is expressed in late G1-phase, peaks during S-phase, and is present until early to mid-mitosis, when it is destroyed by ubiquitin-mediated proteolysis (Cheng et al. 1999; Weinreich and Stillman 1999; Ferreira et al. 2000; Miller et al. 2009), The timing of Dbf4 destruction suggests that Dbf4 has post-replicative functions. Indeed, recent work has shown that Dbf4 prevents premature exit from mitosis and also controls the segregation of homologous chromosomes in meiosis I by a direct interaction with Cdc5, the only Polo-like kinase in budding yeast (Matos et al. 2008; Miller et al. 2009; Chen and Weinreich 2010). Rad53mediated phosphorylation of Dbf4 postpones late origin firing during replication stress (Lopez-Mosqueda et al. 2010; Zegerman and Diffley 2010; Duch et al. 2011) but Cdc7Dbf4 kinase activity is only reduced 2-fold by Rad53-dependent Dbf4 phosphorylation (Weinreich and Stillman 1999). It is clear that Dbf4 is an in vivo target of Rad53 and interacts with Rad53 (Kihara et al. 2000; Duncker et al. 2002; Matthews et al. 2012), but the molecular details of the Rad53-Dbf4 interaction and how Rad53 phosphorylation of Dbf4 prevents late origin activation are unclear. 107 Rad53 is unique in budding yeast in that it contains two FHA (fork-head associated) domains, termed FHA1 and FHA2, which flank a central kinase domain. FHA domains comprise a ubiquitous class of protein-protein interaction modules found in more than 200 different proteins from yeast to mammals (Mahajan et al. 2008). Structural studies show that FHA domains fold into a ß-sandwich composed of 6-stranded and 5-stranded ß sheets (Durocher et al. 2000). Four of the five most conserved residues in the domain are situated in substrate binding loops that selectively recognize a phosphorylated threonine (Liang and Van Doren 2008). Orientated peptide library screening identified consensus phospho-threonine peptides for the FHA1 and FHA2 domains and the structural basis of their interaction with the Rad53 FHA domains were also determined (Liao et al. 1999; Durocher et al. 2000; Byeon et al. 2001). The FHA1 domain preferentially binds peptides containing the consensus sequence pTxxD but the FHA2 domain prefers isoleucine at the +3 position, pTxxI. Here we have mapped the Dbf4 residues required for a physical interaction with Rad53. We found that a short sequence from residues 100-109 that contained a potential FHA1 binding site consensus (T-x-x-E) and the adjacent BRCT (BRCA1 carboxyl-terminal) domain were both required for Rad53 binding. Interestingly, both Rad53 FHA domains were required to bind Dbf4 and depended on a critical threonine 105 residue, which differs from a previous report (Matthews et al. 2012). This suggested that the FHA1 and FHA2 domains bind to the same Dbf4 sequence containing a pT105 residue. Biochemical assays confirmed that the FHA1 domain bound to a Dbf4 pT105-X-X-E peptide in a phosphorylation dependent manner. However the FHA2 domain did not 108 bind the same isolated peptide suggesting that additional contacts with Dbf4 are required for stable binding of the FHA2 domain. Lastly, abrogation of the Rad53-Dbf4 physical interaction blocked Dbf4 phosphorylation by Rad53 and allowed late origin firing in the presence of HU. We suggest that Dbf4 is phosphorylated on T105 and in response to replication fork arrest, the pT105-x-x-E FHA1 binding site together with the BRCT domain cooperate to form a docking site for Rad53. The Rad53 physical interaction then promotes Dbf4 phosphorylation at critical downstream sites to inhibit late origin firing. 109 RESULTS Rad53 interacts with a sequence preceding the Dbf4 BRCT domain Dbf4 is a downstream substrate of the Rad53 kinase in the DNA replication checkpoint (Masai et al. 1999; Weinreich and Stillman 1999; Lopez-Mosqueda et al. 2010; Zegerman and Diffley 2010; Duch et al. 2011). In the presence of HU, Rad53 phosphorylates multiple sites within Dbf4 to inhibit the late origin firing. Our previous study showed that deletion of Dbf4 residues from 66-109 prevented Rad53-mediated Dbf4 phosphorylation in HU (Gabrielse et al. 2006), suggesting that these residues, which are N-terminal to a conserved BRCT domain, played a critical role in the Rad53Dbf4 interaction. We used a two-hybrid assay to map the Rad53 binding site within Dbf4. Using a series of Dbf4 N-terminal truncations we found that a deletion through residue 65 retained the Rad53-Dbf4 interaction (Figure 13A). However, further deletions to residue 109 (just prior to the BRCT domain) or to residue 221 resulted in a complete loss of Rad53 binding. The dbf4-N!221 mutant was still capable of associating with Cdc7 through its C-terminal motifs M and C (Figure 13A and (Ogino et al. 2001; Harkins et al. 2009). In addition, Dbf4 residues 66-227 were sufficient to interact with Rad53 (Figure 13B). Therefore, Dbf4 residues 66-227 contain a separate domain (or domains) that interacts with the Rad53 kinase. These data also indicate that a sequence within Dbf4 residues 65-109, which is poorly conserved among Dbf4 orthologs (Masai and Arai 2000; Gabrielse et al. 2006), is required for the Rad53 interaction. 110 Figure 13. Mapping the interaction between Dbf4 and Rad53 (A) Deletion mutants in otherwise full-length Dbf4 were tested for a two-hybrid interaction with full-length Rad53. 10-fold serial dilutions of saturated cultures were spotted onto SCM-Trp-Leu plates to visualize total cells and SCM-Trp-Leu-His + 2mM 3AT plates to score the two-hybrid interaction. (B) The Dbf4 N-terminal fragment (residues 66-227) was sufficient for the Rad53 interaction and this interaction requires both the FHA domains. (C) Dbf4 residues 100-227 comprised the minimal region for Rad53 FHA1 domain binding. (D) Schematic of the features in Dbf4 are shown, including the Polo-like kinase (Cdc5) binding site, a conserved BRCT domain, motifs M and C, along with a summary of the Dbf4-FHA1 domain interaction. 111 Figure 13. (cont’d) A Dbf4 Bait Total cells Y2H interaction 1-705aa 1-705aa 66-705aa 110-705aa 222-705aa 222-705aa Prey Vector Rad53 Rad53 Rad53 Rad53 Cdc7 Scm/-Trp-Leu Scm/-Trp-Leu-His +0.5 mM 3AT B Dbf4 Bait Total cells Y2H interaction Rad53-FL Prey 66-227aa Rad53 110-227aa Rad53 65-227aa rad53-R70A 66-227aa rad53-R605A 66-227aa Vector Scm/-Trp-Leu Scm/-Trp-Leu-His +2 mM 3AT 112 Figure 13. (cont’d) C Dbf4 Bait Total cells Y2H interaction Prey Vector FHA1 66-227aa FHA1 82-227aa FHA1 94-227aa FHA1 100-227aa FHA1 104-227aa FHA1 108-227aa FHA1 110-227aa FHA1 66-227aa FHA1 66-109aa FHA1 66-150aa FHA1 66-190aa FHA1 GA159LL FHA1 F166A FHA1 W202A FHA1 66-109aa Cdc5-PBD Scm/-Trp-Leu Scm/-Trp-Leu-His +2 mM 3AT 113 Figure 13. (cont’d) D 1 135 179 N 260 309 M 83 704aa Dbf4 C Thr105 Polo 1 656 BRCT 115 88 100 221 227 66 82 ++++ 94 ++++ 100 ++++ 104 110 66 66 - 109 150 - 66 66 Rad53 Interaction ++++ 190 F166A 227 W202A 66 114 - Figure 14. Analysis of FHA domain-Dbf4 interactions including a screen of all T/Y residues in Dbf4 residues 100-227. (A) The indicated substitutions of Dbf4 threonines were assayed for a two-hybrid interaction using Dbf4 N-terminus (66-227) as bait with the Rad53 FHA2 domain by spotting serial dilutions of cultures onto the indicated media to visualize the total number of cells (left) and the two-hybrid interaction (right). (B and C) The indicated Dbf4 tyrosine mutants were assayed for a two-hybrid interaction with the Rad53 FHA1 (B) and FHA2 (C) domains. Although Y127A and Y204A mutants eliminate the binding of both FHA domains, there is no loss of binding by substituting the structurally similar but nonphosphorylatable amino acid, phenylalanine (Y127F and Y204F). (D) Two hybrid interaction data of the Dbf4 N-terminus (66-227) with all remaining FHA domains in the yeast genome, spotted as in (A). Dma1 (pJK135, 137-302aa), Dma2 (pJK137, 246408aa), Dun1 (pJK275, 1-160aa), Far10 (pJK277, 61-227aa), Fhl1 (pJK279, 253400aa), Fkh1 (pJK281, 41-185aa), Fkh2 (pJK287, 1-254aa), Mek1 (pJK283, 1-152aa), Pml1 (pJK289, 54-204), Xrs2 (pJK285, 1-125aa). 115 Figure 14. (cont’d) A Dbf4 Bait Vector WT T105A T114A T131A T138A T157A T163A T168,169A T171A T175A T188A B Dbf4 Bait WT WT Y127A Y127F Y139A Y198A Y204A Y204F Total cells Y2H interaction Prey FHA2 FHA2 FHA2 FHA2 FHA2 FHA2 FHA2 FHA2 FHA2 FHA2 FHA2 FHA2 Scm/-Trp-Leu Scm/-Trp-Leu-His +2 mM 3AT Total cells Y2H interaction Scm/-Trp-Leu Scm/-Trp-Leu-His +2 mM 3AT 116 Prey Vector FHA1 FHA1 FHA1 FHA1 FHA1 FHA1 FHA1 Figure 14. (cont’d) C Dbf4 Bait WT WT Y127A Y127F Y139A Y198A Y204A Y204F Total cells Y2H interaction Scm/-Trp-Leu Scm/-Trp-Leu-His +2 mM 3AT Total cells Y2H interaction Scm/-Trp-Leu Scm/-Trp-Leu-His +2 mM 3AT D Dbf4 Bait WT WT WT WT WT WT WT WT WT WT WT WT Prey Vector FHA2 FHA2 FHA2 FHA2 FHA2 FHA2 FHA2 117 Prey Vector FHA1 Dma1 Dma2 Dun1 Far10 Fhl1 Fkh1 Fkh2 Mek1 Pml1 Xrs2 Both Rad53 FHA domains are required to interact with the Dbf4 N-terminus Although residues 66-227 are sufficient for the interaction with full-length Rad53, residues 110-227 are not (Figure 13B). So, the same residues required for binding Rad53 within full length Dbf4 are also required in this shorter N-terminal Dbf4 construct. Interestingly, both the FHA1 and FHA2 domains are required for Rad53 binding to the Dbf4 N-terminus (Figure 13B) or to full length Dbf4 (not shown). FHA domains are phospho-threonine specific protein binding modules and recognition of the pT residue requires a conserved arginine residue (Durocher et al. 2000; Byeon et al. 2001). Alanine substitutions of the corresponding arginine residues in the FHA1 and FHA2 domains (R70A and R605A, respectively) abolished the Dbf4 interaction (Figure 13B). These results not only indicate that Rad53 binding to the Dbf4 N terminus relies on both FHA domains, but also suggest that the Rad53-Dbf4 interaction is phosphorylationdependent. To identify the FHA binding sites in Dbf4, we first verified that the FHA1 (Figure 13C) and FHA2 (Figure 14A) domains could bind Dbf4 residues 66-227 independently. We then tested a series of deletion constructs within the 66-227 region for their ability to bind FHA1 and FHA2. Although Dbf4 constructs as short as 100-227 retained FHA binding, deletions beyond residue 100 completely lost FHA1 (and FHA2) binding. This indicates that a Dbf4 sequence following residue 100 is required for the FHA domain interactions. Although residues 66-109 preceding the BRCT domain are required for the Rad53 interaction (Figure 13A), residues 66-109 were not sufficient for the interaction with the FHA1 domain (Figure 13C). As a control, Dbf4 residues 66-109 118 were sufficient to interact with the Cdc5 Polo-box domain (PBD) (Figure 13C, bottom) as shown previously (Miller et al. 2009). Finally, the Dbf4-FHA domain interaction also required the Dbf4 BRCT domain comprising residues ~115-224. Any C-terminal deletion that affected the BRCT domain or point mutants in conserved BRCT residues (G159L, A160L, F166L, and W202A) disrupted the Dbf4-FHA domain interaction. To summarize, Dbf4 residues 100-227 comprise the minimal region required to bind Rad53 by a twohybrid assay and mutation of residues within the BRCT domain or immediately preceding it abolish that interaction (Figure 13D). Rad53 FHA domains recognize a T-x-x-E-L motif in the Dbf4 N-terminus In orientated peptide library screens the Rad53 FHA1 and FHA2 domains were shown to selectively bind phospho-threonine (Durocher et al. 2000; Byeon et al. 2001). Therefore, we mutated each threonine to alanine within Dbf4 residues 100-227, i.e. the minimal Rad53 binding region we defined (Figure 14A and Figure 15A). We found that the T105A or T171A substitutions strongly impaired the Dbf4-FHA domain interaction. The surrounding sequences of these two threonines (T105-P-K-E and T171-I-V-I) strongly resemble the binding consensus for FHA1 (pT-x-x-D) and FHA2 domains (pT-x-x-V/L), respectively (Durocher et al. 2000; Byeon et al. 2001). However, a recent crystal structure of the Dbf4 BRCT domain (Matthews et al. 2012), showed that the T171-I-V-I sequence forms part of the hydrophobic core of the BRCT domain and is not solvent accessible (T171 is only partially buried). So although the T171-I-V-I motif conforms to a typical FHA2 binding sequence (pT-x-x-V/L), this motif is buried and is therefore unlikely to interact with the FHA2 domain directly. However, T105 maps just prior to an alpha 119 helix adjacent to the BRCT domain and is solvent accessible. Based on two-hybrid data (described below), we suggest that the Rad53 FHA domains directly recognize T105. We next determined the amino acids required for Rad53 binding between residues 100114 using a series of point mutants. In addition to T105, we found that mutation of V104, E108, L109 and W112 disrupted FHA1 and FHA2 domain binding as summarized (Figure 15B; two-hybrid data in Figure 16). The V104A substitution disrupted the interaction but V104L had only a modest effect, suggesting a structural role or hydrophobic contact for this residue. The E108A mutation strongly impaired FHA binding and E108K abolished FHA binding. However, a conservative E108D mutation retained FHA binding, suggesting that glutamate and aspartate are interchangeable at the +3 position following T105. As expected for an FHA binding consensus site, the Dbf4 residues P106 and K107 at the +1 and +2 positions to T105 were not important for binding, strongly suggesting that T105-x-x-E is a bona fide FHA1 binding site. Our sitedirect mutagenesis studies also found that several hydrophobic residues nearby T105 are important. The loss of interaction caused by the W112A mutation can be rescued by substituting F, a bulky hydrophobic residue, suggesting that W112 plays a structural role for FHA domain binding. Indeed, W112 falls within an alpha helix preceding the BRCT domain and makes hydrophobic contacts with the BRCT domain (Matthews et al. 2012). However, L109 may be directly involved in FHA binding, since it is adjacent to E108 and neither the L109A nor L109V mutants interact with the FHA domains (Figure 15B). 120 Figure 15. The Rad53 FHA domains require a T105-x-x-E-L motif in the Dbf4 N terminus for interaction (A) An alanine scan of all Dbf4 threonines within the minimal Rad53 binding region (residues 100-227) using two-hybrid assays. (B) Summary of Dbf4 mutants within residues 100-114 for their effect on the interaction of FHA1 and FHA2 domains, respectively. The growth assays are shown in Figure 16. 121 Figure 15. (cont’d) A Dbf4 Bait Vector WT T105A T114A T131A T138A T157A T163A T168,169A T171A T175A T188A Total cells Scm/-Trp-Leu Y2H interaction Prey FHA1 FHA1 FHA1 FHA1 FHA1 FHA1 FHA1 FHA1 FHA1 FHA1 FHA1 FHA1 Scm/-Trp-Leu-His +2 mM 3AT 122 Figure 15. (cont’d) B * * * 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 V E P R V T P K E L L E W Q T A A K A A E A L A A A E A K D A V A A A F A A V E P R V T P K E L L E W Q T 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 123 FHA1 interaction ++++ ++++ ++++ ++++ ++++ ++++ ++++ + ++++ + ++++ ++++ ++++ + ++++ + + ++++ ++++ ++++ ++++ ++++ ++++ FHA1 interaction FHA2 interaction ++ ++ ++ ++ ++ ++ ++ ++ ++ ++++ ++++ ++ ++ ++ ++ ++ ++ ++ FHA2 interaction Figure 16. Dbf4 residues V104, T105, E108, L109, and W112 are required for the binding the Rad53 FHA domains The indicated substitutions within residues 100-114 of the Dbf4 N-terminal (66-227) bait plasmid were assayed for a two-hybrid interaction with the Rad53 FHA1 (panel A) and FHA2 domains (panel B). Spotting as in Figure 15. 124 Figure 16. (cont’d) A Dbf4 Bait Vector WT V100A E101A E101K P102A R103A R103E V104A V104L T105A P106A K107E E108A E108K E108D L109A L110A E111A W112A W112F Q113A T114A Total cells Scm/-Trp-Leu Y2H interaction Scm/-Trp-Leu-His +2 mM 3AT 125 Prey FHA1 FHA1 FHA1 FHA1 FHA1 FHA1 FHA1 FHA1 FHA1 FHA1 FHA1 FHA1 FHA1 FHA1 FHA1 FHA1 FHA1 FHA1 FHA1 FHA1 FHA1 FHA1 FHA1 FHA1 Figure 16. (cont’d) B Dbf4 Bait Vector WT V100A E101A E101K P102A R103A R103E V104A V104L T105A P106A K107E E108A E108K E108D L109A L110A E111A W112A W112F Q113A T114A Total cells Scm/-Trp-Leu Y2H interaction Scm/-Trp-Leu-His +2 mM 3AT 126 Prey FHA2 FHA2 FHA2 FHA2 FHA2 FHA2 FHA2 FHA2 FHA2 FHA2 FHA2 FHA2 FHA2 FHA2 FHA2 FHA2 FHA2 FHA2 FHA2 FHA2 FHA2 FHA2 FHA2 FHA2 Figure 17. The Rad53 FHA1 domain directly binds to a T105 phosphorylated Dbf4 peptide (A) Biotinylated Dbf4 peptides (residues 98-113) were tested for interaction with the purified 6His-FHA1 domain using the AlphaScreen Assay. Data represents the average of three independent experiments ± SEM. (B) Purified 6His-FHA2 domain does not interact with the pThr105 Dbf4 peptide, but does selectively bind a Rad9 phosphorylated peptide. (C) The Dbf4-FHA1 domain interaction was competed by nonbiotinylated, T105-phosphorylated Dbf4 peptide (pThr105), a peptide containing the optimal FHA1 binding sequence (pT105-E108D), but not by the T105 (nonphosphorylated) Dbf4 peptide. (D) Summary of peptide sequences and the IC50 values determined by competition assays. 127 Figure 17. (cont’d) AlphaScreen signals (cps x1000) A FHA1-WT FHA1-R70A 80 60 40 20 0 B Thr105 pThr105 Dbf4 peptides Rad53-FHA2 AlphaScreen signals (cps x1000) 150 100 50 0 Thr pThr Dbf4 Thr pThr Rad9 128 Figure 17. (cont’d) Thr105 100 % Maxiaml counts C pThr105 80 pThr105-E108D 60 40 20 0 101 102 103 104 105 106 Non-biotin labeled Dbf4 peptides (nM) 129 Figure 17. (cont’d) D Peptide Dbf4 (98-113aa) pDbf4 (pThr105) pDbf4-V104A pDBf4-E108A pDbf4-E108D pDbf4-L109A Rad9 pRad9 Sequence KNV EPR VTP KEL LEW Q KNV EPR V(pT)P KEL LEW Q KNV EPR A(pT)P KEL LEW Q KNV EPR V(pT)P KAL LEW Q KNV EPR V(pT)P KDL LEW Q KNV EPR V(pT)P KEA LEW Q IMS EVE LTQ ELP EVE IMS EVE L(pT)Q ELP EVE 130 IC50 ( M) >1500 53.639 176.664 422.522 2.285 334.297 The Rad53 FHA2 domain binds peptides with a different binding consensus from the FHA1 domain (Liao et al. 1999; Wang et al. 2000; Byeon et al. 2001). However, using a two-hybrid assay we found that the FHA2 domain interacts with Dbf4 using the same residues as the FHA1 domain, albeit more weakly than the FHA1-Dbf4 interaction. Significant exceptions are that the K107A or K107E substitutions (at the +2 position) substantially enhance FHA2 binding but do not affect FHA1 binding (Figure 15B). Taken together, these results suggest that both the Rad53 FHA1 and FHA2 domains recognize the Dbf4 sequence, T105-x-x-E-L. The Dbf4-FHA1 domain interaction is phospho-threonine dependent To investigate whether the Dbf4-FHA1 domain interaction required phosphorylation of Dbf4 residue T105, we purified the FHA1 domain and tested its ability to bind synthetic Dbf4 peptides using the AlphaScreen proximity assay (Ullman et al. 1994). The FHA1 domain bound to the biotinylated Dbf4 peptides containing residues 98-113 but only if T105 was phosphorylated (Figure 17A). In addition, mutation of the conserved R70 to A in FHA1 abolished the interaction with the Dbf4 pT105 peptide. This data indicates that the Dbf4-FHA1 domain interaction requires T105 phosphorylation. In contrast, although the FHA2 domain bound efficiently to an optimal Rad9 phosphorylated peptide (Byeon et al. 2001) it was unable to bind the same pT105 Dbf4 peptide (Figure 17B). FHA domains bind to pT plus adjacent residues but also make further extensive substrate contacts outside the pT binding loop (Mahajan et al. 2008). Since neither FHA domain bound to Dbf4 residues 66-109 in the two-hybrid assay unless the BRCT domain was 131 included, FHA1 and FHA2 binding to Dbf4 also requires the BRCT domain and includes perhaps distinct FHA-BRCT contacts. To test whether additional residues discovered in the two-hybrid screen were important for the FHA1-Dbf4 peptide interaction, we used non-biotinylated peptides to compete FHA1::biotin-pT105 peptide binding. As expected, the FHA1::biotin-pT105 interaction was competed by an identical pT105 peptide but not by a non-phosphorylated T105 peptide or by an unrelated phospho-peptide (Figure 17C and Figure 18A). The FHA1pT105 peptide competed with an IC50 of 50-60 !M, indicating a moderate FHA1 binding affinity to this peptide. In the yeast two-hybrid assays, we found that E108 and the hydrophobic residues immediately adjacent to the pT105-x-x-E motif were critical for the FHA1 interaction. In agreement with that data, a pT105-x-x A peptide was significantly impaired in its ability to compete the FHA1::biotin-pT105 peptide interaction (Figure 18A). Similarly, alanine substitutions of V104 or L109 within otherwise identical pT105 peptides reduced the ability to compete the FHA1::biotin-pT05 peptide interaction (Figure 18B). Finally, the E108D mutation, which did not affect the Rad53-Dbf4 interaction in the two-hybrid assay and matches the optimal binding sequence for the FHA1 domain, competed the interaction but with a much higher binding affinity (1-5 !M) as shown in Figure 17C. Based on the two-hybrid and biochemical assays, the Rad53 FHA1 domain selectively binds a pT-x-x-E sequence, which closely conforms to an FHA1 binding consensus sequence. 132 Figure 18. Dbf4 residues V104, E108, and L109 are critical for the specific binding of Rad53 FHA domains (A) The Dbf4 biotinylated peptide pThr105-FHA1 interaction was competed by the nonbiotinylated T105-phosphorylated Dbf4 peptides (pThr105), but not by the same Dbf4 peptide with an E108A substitution, or by an unrelated phospho-serine peptide (pSpc72). (B) The pThr105-V104A and pThr105-L109A peptides were also defective in competing the biotinylated pThr105-FHA1 interaction. pThr105 100 % Maxiaml counts A 80 pThr105-E108A 60 pSpc72 (unrelated) 40 20 0 102 103 104 105 106 Non-biotin labeled Dbf4 peptides (nM) pThr105 100 % Maxiaml counts B pThr105-V104A 80 pThr105-L109A 60 40 20 0 102 103 104 105 106 Non-biotin labeled Dbf4 peptides (nM) 133 Figure 19. DDK, Rad53 and Cdc5 form a ternary protein complex HA-Cdc7-Dbf4 complexes were immunoprecipitated from baculovirus-infected Sf9 cells using 12CA5 antibodies and examined for co-immunoprecipitation of Rad53 and 3Myc-Cdc5 by Western blotting. Rad53 and Cdc5 were coimmunoprecipitated by wild-type HA-Cdc7-Dbf4 but not by the HA-Cdc7-Dbf4(N!109) mutant (middle). Following 12CA5 immunoprecipitation, proteins were eluted from the beads using HA peptide and subjected for another round of immunoprecipitation by 9E10 antibodies. Rad53 was co-immunoprecipitated with 3Myc-Cdc5 and wild type DDK but not if Dbf4-N!109 was expressed (right). 134 WT WT WT WT WT WT WT WT Dbf4 WT Figure 19. (cont’d) 3Myc-Cdc5 Rad53 + + + + - + + + + - + + + + + + - + + + + - + + + + + + - + + + + - + + 1 HA-Cdc7 1 1 2 3 4 2 3 4 2 3 4 WB: anti-Dbf4/Cdc7 Dbf4 WB: anti-Myc Cdc5 Cdc7 Rad53 WB: anti-Rad53 IP: HA WCE 135 re-IP: Myc Dbf4 mediates the association of Cdc7, Rad53, and Cdc5 kinases Although Dbf4 is well known for its essential role in binding and activating Cdc7 to initiate DNA replication, we recently proposed that Dbf4 also functions as a molecular scaffold for the Cdc7 and Cdc5 kinases. Dbf4 residues 83-88 directly interact with the Polo-box domain of the Cdc5 kinase and functionally inhibit Cdc5 in the mitotic exit network (MEN) (Miller et al. 2009; Chen and Weinreich 2010). Now that we have defined a distinct binding site for the Rad53 kinase in the Dbf4 N-terminus, in close proximity to the Cdc5 binding site, we wondered whether Dbf4 formed a ternary complex with the Rad53, Cdc5, and Cdc7 kinases. To examine the DDK interaction with Cdc5 and Rad53 we employed a baculovirus system to express Rad53, Cdc5, Cdc7, and various Dbf4 derivatives in Sf9 cells. Consistent with previous reports (Miller et al. 2009; Chen and Weinreich 2010), Cdc5 was co-immunoprecipitated (co-IPd) with wild type HA-Cdc7-Dbf4 but not with the HACdc7-Dbf4(N!109) truncation derivative (Figure 19, middle panel). Similarly, Rad53 bound to wild type HA-Cdc7-Dbf4, but not to HA-Cdc7-Dbf4(N!109). These results indicate that the association of Rad53 and Cdc5 with DDK depends on the first 109 residues of Dbf4, which contains the Cdc5 binding site (residues 83-88) and a Rad53 binding site (residues 104-109). The co-immunoprecipitation results suggest two different possibilities. Either DDK exists in two distinct protein complexes (DDK-Rad53 and DDK-Cdc5) or alternatively, DDK can bind to Cdc5 and Rad53 simultaneously. To clarify this, we asked whether 136 Rad53 binds the DDK-Cdc5 complex by performing a sequential co-IP. We expressed all four proteins in Sf9 cells and IPd DDK using the HA tag on the Cdc7 subunit. This procedure immunoprecipitates proteins bound to DDK, which will include DDK-(MycCdc5), DDK-Rad53 and presumptive DDK-(Myc-Cdc5)-Rad53 complexes. We then eluted the bound proteins using 1 mM HA peptide and performed a second round of immunoprecipitation using 9E10 monoclonal antibodies to IP just the DDK-(Myc-Cdc5) complexes. Rad53 was present in the second IP (Figure 19, right panel) indicating that Rad53 forms a ternary complex with Cdc5 and DDK. Together these results demonstrate that the Dbf4 N -terminus acts as a docking site for both Rad53 and Cdc5, and that both kinases can simultaneously associate with DDK. A Rad53 checkpoint defect together with loss of specific Dbf4 N-terminal residues results in synthetic lethality DDK and rad53 mutants show a series of complex genetic interactions. For instance, hypomorphic cdc7 and dbf4 mutants are synthetically lethal with rad53 hypomorphic mutants (Desany et al. 1998; Dohrmann et al. 1999; Dohrmann and Sclafani 2006; Gabrielse et al. 2006). However, cdc7-1 is also synthetically lethal with the rad53-31 mutant, which is checkpoint proficient (Dohrmann et al. 1999). We previously reported that the dbf4-N!109 mutant was synthetically lethal with the rad53-1 hypomorphic mutant (Gabrielse et al. 2006). This is interesting since the dbf4-N!109 mutant exhibits an apparently normal S-phase, is not defective for activating early or late replication origins, and is not sensitive to genotoxic agents (Gabrielse et al. 2006). However, the Dbf4-N!109 protein is defective for binding Cdc5 (Miller et al. 2009; Chen and 137 Weinreich 2010) and Rad53 (this study). Therefore, we tested whether the synthetic lethality between dbf4-N!109 and rad53-1 was due to the loss of the Dbf4-Cdc5 or Dbf4-Rad53 interactions. We first sequenced the rad53-1 gene (Weinert et al. 1994) and found a single G653E point mutation, which is identical to that reported for the rad53-11 allele (Dohrmann and Sclafani 2006). G653 falls within a loop between the b6 and b7 strands of the FHA2 domain and is adjacent to the conserved N655 residue, which plays an important role in substrate recognition (Figure 20A) (Byeon et al. 2001). The rad53-1 (G653E) or N655A full-length Rad53 mutants were unable to bind the Dbf4 N-terminus in the two-hybrid assay (Figure 20B), highlighting the importance of the FHA2 domain for the Dbf4 interaction. Both mutants were expressed similarly to the wild type (data not shown). Similarly we found that the rad53-R70A mutant, which cannot interact with Dbf4, was synthetically sick or lethal with dbf4-N!109 but obviously not with DBF4 (Figure 20C). Not surprisingly, we also observed synthetic lethality between dbf4-N!109 and the rad53-K227A (kinase dead) allele (Figure 20D). Since the rad53R70A and rad53-G653E (rad53-1) mutants are defective for interacting with DDK to begin with, the synthetic lethality with dbf4-N!109 cannot be due to the further loss of just the Rad53 binding site on Dbf4. The synthetic lethality is likely caused by compromised Rad53 function coupled with loss of a Rad53-independent function of Dbf4 present in the N-terminal 109 residues. We know that this function is not the ability to bind Cdc5, since a dbf4-!82-88 mutant, which is completely defective for binding Cdc5 (Miller et al. 2009; Chen and Weinreich 2010), is not synthetically lethal with rad53-1 (Figure 21A). Furthermore, a rad53! sml1! strain is not synthetically lethal with the dbf4-!82-88 allele (defective for Cdc5 binding) or the dbf4-!100-109 allele 138 (defective for Rad53 binding) but is synthetically lethal with the dbf4-N!109 allele (Figure 21B). Deletion analysis of the region between residues 65 and 109 indicates that loss of sequences between residues 82-109 causes synthetic lethality with rad53! sml1! (Figure 21), which includes both the Cdc5 and Rad53 binding sites. This genetic data strongly suggest (remarkably) that yet another function of the Dbf4 N-terminus cooperates with Rad53 to ensure cell viability. The Dbf4-Rad53 physical interaction is required to inhibit late origin firing during replication checkpoint activation In response to replication fork arrest, Rad53 phosphorylates both Dbf4 and Sld3 to inhibit late origin firing (Lopez-Mosqueda et al. 2010; Zegerman and Diffley 2010). The critical Rad53 phosphorylation sites on Dbf4 map toward the C-terminus between motifs M and C. A dbf4-4A mutant that changes 4 serine and threonine Rad53 phosphorylation sites to alanine is sufficient to allow late origin activation when combined with an sld3-38A mutant, containing alanine mutations in 38 Rad53 phosphorylation sites (Zegerman and Diffley 2010). We hypothesized that Rad53 phosphorylation of Dbf4 depends on its physical interaction with the Dbf4 N-terminus. To test this, we examined whether the combination of a dbf4-N!109 mutant (defective for Rad53 binding) and the sld3-38A mutant, which cannot be phosphorylated by Rad53, would allow late origin firing in the presence of HU. Yeast cells were synchronized in G1 phase using mating pheromone and then released into S phase in the presence of 0.2 M HU to stall replication forks from early origins. At different time points following release from the G1 arrest, replication intermediates (RI) near ARSs were separated on 139 alkaline gels and detected by Southern blotting with ARS-specific probes to measure replication origin activity. As a control, Rad53 was activated (evidenced by the phosphorylation-dependent mobility shift) in both wild type and mutant cells following HU treatment (Figure 22A), indicating that neither the dbf4 nor the sld3 mutations affect Rad53 checkpoint activation. The early origin, ARS305, was active in the wild type, dbf4-N!109, dbf4-4A sld3-38A, and dbf4-N!109 sld3-38A mutant strains indicating that induction of the replication checkpoint does not interfere with early origin firing in these cells. Although Rad53 activation inhibited the firing of late origins ARS501 and ARS603 in the wild type and the single mutants as expected (Lopez-Mosqueda et al. 2010; Zegerman and Diffley 2010; Duch et al. 2011), replication intermediates were detected at late origins in both the sld3-38A dbf4-4A and sld3-38A dbf4-N!109 double mutants to a similar extent (Figure 22C and D). Thus, the dbf4-N!109 mutant defective for Dbf4-Rad53 binding is similarly defective in preventing late origin firing in HU as the dbf4-4A phosphorylation site allele. This result is consistent with the previous observation that the dbf4-N!109 mutant significantly impaired Rad53-mediated Dbf4 hyper-phosphorylation in HU (Gabrielse et al. 2006). These data strongly suggest that Rad53 must stably interact with Dbf4 through its N-terminal binding site to phosphorylate Dbf4 and inhibit late origin firing in response to HU. 140 Figure 20. dbf4-N!109 is synthetically lethal with rad53-R70A, rad53-K227A, and rad53-G653E (A) Sequence alignment of all FHA domain-containing proteins in Saccharomyces cerevisiae. Absolutely conserved residues are highlighted in aquamarine. The Rad53 R70 and R605 residues are marked below with a black circle. The rad53-1 mutant was sequenced and found to contain a single point mutation (G653E) within the ß 6-7 loop of the FHA2 domain (highlighted yellow, black square). (B) The rad53-G653E and rad53-N655A mutants do not interact with Dbf4 in yeast two-hybrid assays. (C and D) Representative tetrads from diploid strains of genotype DBF4/dbf4-N!109 RAD53/rad53-R70A and DBF4/dbf4-N!109 RAD53/rad53-K227A were sporulated and dissected onto YPD plates. Recombinant genotypes are indicated. For interpretation of the references to color in this and all other figures, the reader is referred to the electronic version of this dissertation. 141 Figure 20. (cont’d) 142 Figure 20. (cont’d) B Dbf4 Rad53 Prey Vector Dbf4 Full-length WT Dbf4 G653E (rad53-1) Dbf4 T654A Dbf4 N655A Bait Total cells Scm/-Trp-Leu Y2H interaction Scm/-Trp-Leu-His +2 mM 3AT D C rad53-R70A rad53-K227A rad53-R70A rad53-K227A 143 Figure 21. The synthetic lethality between dbf4-N!109 and rad53-1 or rad53! is not due solely to either loss of Cdc5 interaction or increased Dbf4 stability but requires sequences between residues 82-109 Wild type and various dbf4 mutants were cloned in low-copy number (ARS/CEN/LEU2) vectors, driven by the DBF4 endogenous promoter. Plasmids were transformed into M1589 (rad53-1 dbf4!::kanMX6 [pDBF4-URA3]) or M3581 (rad53!::TRP1 sml1!::HIS3 dbf4!::kanMX6 [pDBF4-URA3]) and the wild-type DBF4-URA3 plasmids were selected against on FOA. Cells that could not grow on FOA plates were scored as having a synthetic lethal interaction. The N!65 deletion causes increased Dbf4 stability by deleting sequences important for ubiquitin-mediated proteolysis. The !82-88 deletion prevents Cdc5 binding to Dbf4, while the !100-109 deletion prevents Rad53 binding to Dbf4. 144 Figure 21. (cont’d) A B Total cells Y2H interaction Scm/-Trp-Leu Scm/-Trp-Leu-His +0.5 mM 3AT Total cells Y2H interaction Scm/-Trp-Leu Full-length Dbf4 Bait WT Prey Scm/-Trp-Leu-His Prey Cdc5-PBD Full-length Rad53 Cdc5-PBD Full-length Rad53 Full-length Dbf4 Bait WT Vector Vector 145 Figure 22. The dbf4-N!109 sld3-38A double mutant allows late origin firing in the presence of HU (A) Wild type and mutant cells were synchronized in G1 phase by alpha-factor and released into S phase into medium containing 0.2 mM HU for the indicated times. Total protein extracts were examined by Western blotting for Rad53 to assess Rad53 activation (upper band). (B-D) Replication intermediates (RI) were separated by alkaline gel electrophoresis and detected by Southern blotting to measure the activity of early (ARS305) and late (ARS501 and ARS603) origins. Flow cytometry assays and budding index are shown in (E) and (F). 146 Figure 22. (cont’d) WB: anti-Rad53 A sld3-38A 0 30 60 90 120 0 30 60 90 120 0 30 60 90 120 0 30 60 90 120 WT dbf4-4A sld3-38A sld3-38A 0 30 60 90 120 0 30 60 90 120 0 30 60 90 120 0 30 60 90 120 RI SB: ARS305 (Early) B WT dbf4-4A sld3-38A 147 Figure 22. (cont’d) C WT dbf4-4A sld3-38A sld3-38A RI SB: ARS501 (Late) 0 30 60 90 120 0 30 60 90 120 0 30 60 90 120 0 30 60 90 120 148 Figure 22. (cont’d) D WT dbf4-4A sld3-38A sld3-38A RI SB: ARS603 (Late) 0 30 60 90 120 0 30 60 90 120 0 30 60 90 120 0 30 60 90 120 149 Figure 22. (cont’d) E dbf4-4A sld3-38A Time (mins) after release from G1 to HU WT sld3-38A 120 90 60 30 0 Asyn 1C 2C 1C 2C 1C F Budding index (%) 100 80 WT 60 dbf4-4A sld3-38A 40 20 0 0 30 60 90 120 Time (mins) after release from G1 into HU 150 2C 1C 2C DISCUSSION Rad53 interacts with Dbf4 using a phospho-threonine dependent mechanism Multiple groups have reported genetic and physical interactions between S. cerevisiae Dbf4 and Rad53 (Dohrmann et al. 1999; Weinreich and Stillman 1999; Kihara et al. 2000; Duncker et al. 2002; Gabrielse et al. 2006; Matthews et al. 2012), and S. pombe Dfp1 and Cds1 (Takeda et al. 2001; Fung et al. 2002). Furthermore, in response to DNA damage, human and Xenopus DDK are downstream targets of ATR (Costanzo et al. 2003; Lee et al. 2012). The Dbf4-Rad53 interaction is conserved in yeasts and a DDKATR interaction in vertebrates because it likely promotes genome stability. Here we have mapped a Rad53 binding site in the Dbf4 N-terminus and have shown that a Rad53-Dbf4 physical interaction is critical for regulating late replication origin firing. The minimal Rad53 binding region corresponds to Dbf4 residues 100-227, which comprise the Dbf4 BRCT domain (~118-224) and residues immediately N-terminal to this domain. Mutations in either conserved BRCT residues or residues 100-109 abrogated Rad53 binding. This indicates that the BRCT domain and the region preceding it are both required for Rad53 binding. Surprisingly, and despite their different consensus peptide binding sites, both Rad53 FHA domains interacted with this 100-109 region independently and apparently using the same Dbf4 residues (see below). Furthermore, since mutations that impair phospho-threonine substrate recognition in either Rad53 FHA domain blocked the interaction with Dbf4, this further suggested that the Rad53 interaction with Dbf4 is mediated by phosphorylation and is multivalent. 151 Very recently a crystal structure of the Dbf4 BRCT domain (that included residues 98 to 221) was described (Matthews et al. 2012). These authors also showed that Rad53 interacted with the Dbf4 BRCT domain plus the preceding alpha helix using two-hybrid assays, however none of threonines contained within the structure was shown to directly interact with Rad53. So a phosphorylation-independent interaction between Dbf4 and Rad53 was proposed. In contrast, our studies showed that residues T105, E108, L109, W112, and R209 (not shown) mediated the Dbf4-Rad53 interaction. The structure of the Dbf4 N-terminus allows us to rationalize this data (data not shown). T105 is solvent exposed and occurs within a sequence (T-x-x-E) that closely matches a FHA1 binding site. The E108 residue has the same spatial orientation as T105, and L109 is directly adjacent to E108. The W112 residue packs against L214 present at the C-terminus of the BRCT a3 helix. This hydrophobic interaction presumably helps stabilize the a0-a3 orientation and would explain why a W112A mutation disrupts the Dbf4-Rad53 interaction but W112F does not. Finally, R209 on the a3 helix is solvent exposed and is suitably oriented to interact with an FHA domain bound to a0 or, alternatively, to mediate BRCT-BRCT domain interactions. In a tandem BRCT-BRCT dimer the a2 helix from one monomer packs against the a1 and a3 helices from the second monomer (Glover et al. 2004). Mutation of R209 but not K212 (which is oriented orthogonally to R209, away from a0) abolished the Dbf4-FHA1 two-hybrid interaction (JK and MW, unpublished). Since the purified Rad53 FHA1 domain bound only to T105 phosphorylated Dbf4 peptides, together these data strongly suggest a phospho-threonine dependent binding mechanism between Dbf4 and the Rad53 FHA1 152 in which the pT105-x-x-E-L motif in Dbf4 mediates a direct interaction with the Rad53 FHA domains. The FHA1 binding site in Dbf4 is pT-x-x-E, instead of the preferred pT-x-x-D. A pT-x-x-D Dbf4 peptide (that substituted D108 for E) bound to the FHA1 domain significantly better than the wild type but gave a similar two-hybrid interaction with Rad53. The use of a lower affinity pT-x-x-E interaction may reflect a selection for some biological property needed for the Rad53-Dbf4 interaction. Alternatively, a high-affinity binding site might not be required since the Dbf4 BRCT domain also contributes to Rad53 binding as shown by our study and also an earlier study (Duncker et al. 2002). In summary, we have identified pT105-x-x-E as a Dbf4 motif that binds to the Rad53 FHA1 domain. Although the Rad53 FHA2 domain binds to the same sequence in the two-hybrid assay (Figure 15), it does not bind to the pT-x-x-E peptide in vitro and this sequence does not match the optimal FHA2 binding site consensus. Although the FHA2 domain might bind to Dbf4 indirectly in the two-hybrid assay, the FHA2 interaction still occurs in a strain deleted for Rad53 (data not shown) and so, it is not mediated by endogenous Rad53. Interestingly, no other FHA domain in yeast can bind to this Dbf4 sequence (Figure 14D), suggesting again that the FHA2 interaction is biologically relevant. Since a previous study also demonstrated an interaction between FHA2 and Dbf4 using GSTpull downs from yeast (Duncker et al. 2002), we suggest that the FHA2 domain interaction with Dbf4 is stabilized in vivo by additional contacts within the BRCT domain or by the FHA1-pT105 interaction. 153 Models for Rad53 binding to Dbf4 We propose two models to explain how Rad53 interacts with Dbf4. Either Rad53 uses each FHA domain to bind two separate sites within Dbf4 or both Rad53 FHA domains bind to the same pT-x-x-E sequence, but on different Dbf4 subunits within a Dbf4 multimer. FHA1 and FHA2 domains bind to different sites in Dbf4 Our data showed that the FHA1 or FHA2 domain alone could bind the Dbf4 N-terminus (Figure 15). Furthermore, mutation of either FHA domain alone within the context of full length Rad53 abrogated the Rad53-Dbf4 interaction (Figures 1 and 5) indicating that both FHA domains are required for the Dbf4 interaction. Although we have shown that both Rad53 FHA domains require the same T-x-x-E sequence for binding, the BRCT domain may contain another surface for the interaction with full-length Rad53. It is possible that the Rad53 FHA1 domain binds to pT105-x-x-E and that the FHA2 domain binds to another phosphorylated residue in the BRCT domain. This seems unlikely however, since mutation of every other threonine (Figure 15) or tyrosine residue (Figure 14) in the BRCT domain had no effect on FHA1 or FHA2 binding, with the exception of T171 discussed above. Although FHA domains are well known as phospho-threonine binding modules, work from Tsai’s group demonstrated that the Rad53 FHA2 domain can bind both phospho-threonine and phospho-tyrosine containing peptides (Liao et al. 1999; Wang et al. 2000; Byeon et al. 2001). It is still possible that the FHA2 domain binds weakly to a non-consensus site within the BRCT domain and this is not readily detected by our two-hybrid assay. As stated above, it is formally possible that FHA2 154 binds to a bridging protein in the 2-hybrid assay that also recognizes T-x-x-E, but the putative bridging protein is not Rad53 itself. Two FHA domains binding to separate sites would likely promote a higher binding affinity between Rad53 and Dbf4. This may explain why we detected a moderate binding affinity to pT-x-x-E versus pT-x-x-D peptides in our in vitro FHA1 binding assays. Rad53 FHA domains bind to a Dbf4 dimer using the same sequence Given that the Dbf4 residues T105 and E108 were critical to binding both FHA1 and FHA2 domains in the two-hybrid assay, we prefer a second model in which the Dbf4 Ntermini form a dimer using the BRCT domains, and then this Dbf4 dimer provides two pT105-x-x-E108 sites for the binding of the FHA1 and FHA2 domains separately. DDK or Dbf4 oligomerization has been suggested previously (Shellman et al. 1998; Matthews et al. 2012). Furthermore, tandem BRCT domains are present in many proteins where they form dimers that also function as phospho-recognition motifs (Caldecott 2003; Rodriguez and Songyang 2008). In addition, inter-molecular dimerization between BRCT domains has been described for the DNA repair proteins XRCC1 and Ligase III (Cuneo et al. 2011). In support of this model, we saw an interaction between Dbf4 Ntermini using the yeast two-hybrid assay and substitutions of conserved residues in the Dbf4 BRCT domain disrupted the Dbf4-Dbf4 two-hybrid interaction (Figure 23). Although Dbf4-Dbf4 two-hybrid signal was significant, it was a relatively weak interaction compared with the Dbf4-Rad53 interaction. However, the association with Rad53, Cdc7, or other proteins may stabilize Dbf4 dimerization. Arguing against this model is the lack of biochemical data supporting an interaction between the Rad53 155 FHA2 domain and the pT105-x-x-E peptide, however as stated above, FHA2 likely makes additional contacts with the BRCT domain. Dbf4-Rad53 binding is critical for regulation of late origin activation The DNA replication checkpoint is important for controlling the fidelity of DNA synthesis. Upon sensing DNA damage or replication fork stalling, Rad53 activation directly phosphorylates Dbf4 and Sld3, and consequently inhibits late origin firing (LopezMosqueda et al. 2010; Zegerman and Diffley 2010). A dbf4-4A sld3-38A double mutant in the Dbf4 and Sld3 Rad53 phosphorylation sites bypasses the replication checkpoint to allow unchecked S phase progression during replication stress. Similarly, we demonstrated that a dbf4-N!109 mutant defective in the Dbf4-Rad53 interaction coupled with the sld3-38A mutant allows late origin firing in the presence of HU (Figure 22). This indicates that the Rad53-mediated Dbf4 phosphorylation during replication checkpoint likely depends on the physical interaction between Dbf4 and Rad53. We showed previously that deletion of the Dbf4 N-terminal 109 residues largely blocked Rad53-dependent phosphorylation in HU assayed by a Dbf4 mobility shift (Gabrielse et al. 2006). Our study also suggests that the Dbf4-Rad53 interaction is promoted by phosphorylation of Dbf4 residue T105. We propose that the regulation of DDK in the replication checkpoint depends on two phosphorylation events: the first is the phosphorylation of Dbf4 residue T105 by an unknown kinase, which promotes the Rad53-DDK interaction. The second is the subsequent Rad53-mediated phosphorylation of Dbf4 at the critical sites between motifs 156 M and C. Since Rad53 cannot bind Dbf4-N!109 and this leads to a substantial defect in Rad53 phosphorylation of Dbf4, our data implies that stable binding of Rad53 to its targets may be needed for efficient phosphorylation. This is similar for instance to DDK itself, which is targeted to Mcm4 through an N-terminal sequence (Sheu and Stillman 2010). We note that T105 phosphorylation is likely not essential for the Rad53-Dbf4 physical interaction since a Dbf4 quadruple mutant protein (S84A S92A T95A T105A) still underwent a Rad53-dependent shift in HU (Gabrielse et al. 2006). However, since Rad53 interacts with Dbf4 using multiple residues and perhaps two different binding sites, a single point mutation is unlikely to eliminate binding. Role for a DDK-Rad53-Cdc5 complex? We demonstrated that three essential kinases, Rad53, Cdc7 and Cdc5 can form a complex with Dbf4 following co-expression in insect cells. This ternary interaction depends on the N-terminal 109 residues of Dbf4 in agreement with the yeast two-hybrid studies. Although we showed that the Dbf4 interaction with Cdc5 inhibited Cdc5 activation of the MEN pathway (Miller et al. 2009), Cdc5 may have additional functions in a DDK-Rad53-Cdc5 complex. Recent studies show that Cdc5 attenuates Rad53 activation to allow checkpoint adaptation (Donnianni et al. 2010; Vidanes et al. 2010). It is possible that Cdc5 phosphorylates Rad53 with the ternary complex to attenuate its activity. Also, a complex regulation may underlie the DDK interaction with Rad53 and Cdc5. Our two-hybrid results showed that the loss of the Dbf4-Cdc5 interaction (either by deleting residues 82-88, or point mutations within the Cdc5 binding site) promoted a stronger Dbf4-Rad53 two-hybrid interaction (Figure 22). In contrast, Rad53 157 phosphorylates Dbf4 on residue S84 following exposure to HU (Duch et al. 2011). Serine 84 falls within the Cdc5 binding site and we previously demonstrated that phosphorylation of S84 peptide prevented binding to Cdc5 (Chen and Weinreich 2010). This suggests that activated Rad53 might prevent Cdc5 binding to Dbf4 through S84 phosphorylation. Since Cdc5 is expressed late in S phase in contrast to DDK and Rad53, it may only bind DDK after replication is completed. Whether Cdc5 associates with DDK singularly or within a DDK-Cdc5-Rad53 ternary complex in response to replication checkpoint activation (or at other cell cycle transitions) remains to be determined. 158 Figure 23. Evidence for a Dbf4-Dbf4 N-terminal interaction (A-B) Dbf4 N-terminal residues 66-227 were cloned in two-hybrid bait and prey plasmids separately to examine Dbf4 dimerization. Two-hybrid interactions were quantitated by spotting assays on selective media (panel A) or by "-galactosidase assays (panel B). A W202A substitution in the Dbf4 BRCT domain abolished the Dbf4-Dbf4 interaction. (C) The expression of representative Dbf4 mutants in two-hybrid assays is shown by Western blotting against the c-Myc epitope tag on the Gal4BD (DNA Binding Domain) fusions. Whole cell extracts prepared by TCA extraction method were equally loaded onto each lane (Ponceau S staining, left). Gal4BD fused Dbf4 were detected by antiMyc antibody (9E10), followed by anti-mouse second antibody (right). 159 Figure 23. (cont’d) A Bait Total cells Y2H interaction Prey Dbf4-N Vector Dbf4-N Dbf4-N Dbf4-N-W202A Dbf4-N Scm/-Trp-Leu Scm/-Trp-Leu-His +2 mM 3AT Unit of beta-galactosidase B 200 p < 0.001 150 100 50 0 Bait Dbf4-N Dbf4-N Prey vector Dbf4-N 160 C kDa 200 Ponceau S Staining 161 Gal4BD Gal4BD-66-227aa (G159Q) Gal4BD-66-227aa (F166A) Gal4BD-66-227aa (F165A) Gal4BD-66-227aa (GA159LL) Gal4BD-66-227aa (W202A) Gal4BD-66-227aa (W202E) Gal4BD-66-190aa Gal4BD-Dbf4-66-227aa Gal4BD Gal4BD-66-227aa (G159Q) Gal4BD-66-227aa (F166A) Gal4BD-66-227aa (F165A) Gal4BD-66-227aa (GA159LL) Gal4BD-66-227aa (W202A) Gal4BD-66-227aa (W202E) Gal4BD-66-190aa Gal4BD-Dbf4-66-227aa Figure 23. (cont’d) 97 66 45 Gal4BD-Dbf4 (66-227aa) 31 Western Blot (anti-Gal4BD) Figure 24. Sequences between Dbf4 residues 65-88 act to inhibit the Rad53 interaction (A) A series of full-length Dbf4 deletions was assayed by two-hybrid for interaction with full length Rad53 (panel A) or the Cdc5 Polo-box domain (PBD) (panel B). The dbf4!100-109 deletion causes a loss of Rad53 binding, but still allows interaction with the Cdc5-PBD. In particular, the dbf4-R83E and dbf4-!82-88 mutants that cannot bind the PBD domain show increased interaction with Rad53 compared with wild type Dbf4, suggesting that Cdc5 binding can inhibit Rad53 binding to Dbf4. 162 Figure 24. (cont’d) A Total cells Synthetic interaction Scm/-Leu FOA Total cells Synthetic interaction Scm/-Leu FOA Vector DBF4 DBF4 WT B DBF4 Vector WT 163 Figure 25. Dbf4 T105 residue is critical for the Dbf4-FHA1 domain interaction (A) The dbf4-!100-109, dbf4-T105A and dbf4-N!109 mutants cause a loss of FHA1 domain binding in two-hybrid assays. The dbf4-S84A, -S92A, and -T95A mutants did not show any effect on FHA1 domain binding. (B) Substitution of T105A on various Dbf4 truncations consistently caused a loss of interaction with the FHA1 domain. 164 Figure 25. (cont’d) A Dbf4 Bait Total cells Y2H interaction Prey FHA1 Rad53-FHA1 Vector WT Scm/-Trp-Leu Scm/-Trp-Leu-His +2 mM 3AT B Dbf4 Bait 88-227aa 94-227aa 100-227aa Y2H interaction Prey WT Rad53-FHA1 66-227aa Total cells WT WT WT Scm/-Trp-Leu 165 Scm/-Trp-Leu-His +2 mM 3AT MATERIALS AND METHODS Construction of Yeast Strains, Plasmids, and Baculoviruses Plasmids and yeast strains used in this study are listed in Tables 1 and 2. PJ69–4a cells (MATa trp1-901 leu2-3,112 ura3-52 his3-200 gal4! gal80! LYS2::GAL1-HIS3 GAL2ADE2 met::GAL7-lacZ) were used for two-hybrid experiments. All other strains were derivatives of W303-1A (MATa ade2-1 trp1-1 can1-100 leu2-3, 112 his3-11, 15 ura3). The natMX4 cassette flanked with DBF4 target sequences was PCR amplified from p4339 with primers (5’-CTA TCA ACG GCA ATG TTA TTG AAT CAC TTT CTC ATT CAC CCT TGT ACA TGG AGG CCC AGA ATA CC-3’) and (5’- ATG CAA TTG ATA ATA TAT GGA CGA GTA AAT AAG AGT TAA GTC AAT CAG TAT AGC GAC CAG CAT TC-3’) (Goldstein and McCusker 1999), and transformed into M1261 (dbf4-N!109). clonNAT (Werner Bioagents) resistant transformants were confirmed with natMX4 marker and then backcrossed to W303. The epitope-tagged RAD53 strains were made by the method of Longtine et al (Longtine et al. 1998). Deletions and point mutations within DBF4 and RAD53 were generated by site-directed mutagenesis using the QuikChange system (Stratagene). PCR-amplified EcoRI-PstI fragments containing the full-length RAD53 coding sequence (1 to 821), FHA1 domain (1 to 300), FHA2 domain (483 to 821) and DBF4 coding sequence (66 to 227) were cloned into the same sites of pGAD-C1 (Clontech) to give the Gal4 activation domain fusions. Rad53 residues 2-164 were cloned on a BamHI-XhoI into pET24a-GST for expression of His6-GST-FHA1 domain. Construction of baculovirus plasmids encoding wild type Dbf4, Dbf4-N!109, HA-Cdc7, and 3Myc-Cdc5 was previously described (Gabrielse et al. 2006). An NcoIPstI fragment containing the full-length RAD53 coding sequence (1 to 821) was cloned 166 in the baculovirus transfer vector, pAcSG2. High-titer baculoviruses were generated by transfection of Sf9 cells using the BaculoGold kit (BD Biosciences) followed by plaque purification and virus amplification. Growth Conditions, Cell Cycle Synchronization, and Replication intermediate assays Yeast cells were cultured in YPD and Synthetic complete medium (Scm). Cells were synchronized in G1 phase with 5 µg/ml "-factor for 3 hours and released into 0.2M hydroxyurea for the indicated times. The alkaline gel electrophoresis and probes for the replication origins (ARS305, ARS501, ARS603) were previously described (Mantiero et al. 2011). DNA content was analyzed by flow cytometry as previously published (Mantiero et al. 2011). Two-hybrid Analysis Various DBF4 bait constructs containing Gal4 DNA binding domain were transformed with Gal4 activation domain prey plasmids in PJ69–4a and selected on SCM plates lacking tryptophan and leucine. These were spotted at 10-fold serial dilutions on the same plates and also on plates also lacking histidine but containing 2 mm 3aminotriazole and cultured for 2–3 days at 30 °C. #-nitrophenyl-$-D-galactoside (ONPG) (Sigma) $-galactosidase assay was previously described (J. H. Miller (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press). Immunoprecipitation from Sf9 Cells and Western Blotting 167 Sf9 cells were co-infected with HA-Cdc7, 3Myc-Cdc5, Rad53, and Dbf4 mutants as previously described (Chen and Weinreich 2010). Whole cell extracts and IPs were probed with polyclonal antibodies against Cdc7 (1:4000) and Dbf4 (1:1000). Rad53 and 3Myc-Cdc5 were detected with yC-19 (Santa Cruz Biotechnology) and 9E10 antibodies respectively. Protein Purification and Peptide Binding Assays His6-GST-FHA1 and His6-GST-FHA2 domains were induced in BL21(DE3) cells for 3 h at 30 °C using 0.5 mM isopropyl 1-thio-$-d-galactopyranoside. Protein purification and the AlphaScreen luminescence proximity assay (PerkinElmer Life Sciences) were previously described (Chen and Weinreich 2010). All peptides used in this study are listed in Table 6. 168 Table 4. Plasmids Plasmid p4339 pAcSG2 pCG10 pCG40 pCG44 pCG52 pCG53 pCG60 pCG63 pCG64 pCG74 pCG75 pCG91 pCG101 pCG108 pCG110 pCG146 pCG265 pCM16 pCM21 pET24a-GST pGAD-C1 pGAD-Cdc5.3 pGBKT7 pJK18 pJK20 pJK22 pJK25 pJK26 pJK27 pJK29 pJK31 pJK33 pJK34 pJK36 pJK37 pJK39 pJK41 pJK45 pJK47 Description pCRII-TOPO::natRMX4 pRS415-DBF4N!109 pAcSG2-DBF4N!109 pAcSG2-DBF4N!221 pGBKT7-DBF466-227 pYJ204-DBF4N!65 pCG52ADH1 promoter-!(-732)-(-802) pCG60 W202E pCG60 W202A pYJ204-DBF4N!109 pYJ204-DBF4N!221 pAcSG2-DBF4N!65 pCG60 GA159,160LL pCG60 F165A pCG60 F166A pCG60 G159Q pGAD-C1-CDC71-507 pAcSG2-3myc-CDC565-705 pCG60-DBF466-109 pGAD-C1-CDC5421-705 pCG60 T171E pCG60 E108A pCG60 T171S pCG60 V100A pCG60 R103A pCG60 V104A pCG60 P106A pCG60 L109A pCG60 K107A pCG60 T105A E108A pCG60 E108K pCG60 T171A pCG60 E101A pCG60 P102A pYJ204-DBF4N!81 pYJ204-DBF4N!93 169 Source Goldstein and McCusker, 1999 BD Biosciences Gabrielse et al., 2006 Miller et al., 2009 Gabrielse et al., 2006 Miller et al., 2009 Miller et al., 2009 Miller et al., 2009 This study This study Miller et al., 2009 Miller et al., 2009 Gabrielse et al., 2006 This study This study This study This study Harkins et al., 2009 Miller et al., 2009 Miller et al., 2009 Chen and Weinreich, 2010 James et al. 1996 Miller et al., 2009 Clontech This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study Table 4. (cont’d) pJK48 pJK49 pJK51 pJK53 pJK55 pJK57 pJK59 pJK61 pJK67 pJK76 pJK82 pJK83 pJK85 pJK86 pJK89 pJK91 pJK93 pJK95 pJK97 pJK99 pJK101 pJK103 pJK105 pJK107 pJK108 pJK110 pJK112 pJK114 pJK121 pJK122 pJK124 pJK125 pJK126 pJK128 pJK135 pJK137 pJK149 pJK169 pJK170 pJK171 pJK179 pYJ204-DBF4N!99 pCG60 T105S pCG60 K107E pCG60 T131A pCG60 L110A pCG60 E111A pCG60 W112A pCG60 T114A pCG60-DBF4!94-99 pYJ204-DBF4N!88 pCG60 V104L pCG60 L109V pCG60 W112F pCG60 T188A pCG60 T157A pCG60 T163A pCG60 TT168,169AA pCG60 T175A pYJ319 G653E pYJ319 T654A pYJ319 N655A pYJ380 G653E pYJ380 T654A pYJ380 N655A pCG60 Y127A pCG60 Y139A pCG60 Y198A pCG60 Y204A pCG60 Y127S pCG60 Y127T pCG60 I130A pCG60 T171V pCG60 Y204F pCG60 Y127F pGAD-C1-DMA1137-302 pGAD-C1-DMA2246-408 pCG60 T95A pET24a-GST-RAD532-164 pET24a-GST-RAD532-175 pET24a-GST-RAD532-279 pCG60-DBF4N!87 T105A 170 This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study Chen and Weinreich, 2010 This study This study This study This study Table 4. (cont’d) pJK181 pJK185 pJK269 pJK275 pJK277 pJK279 pJK281 pJK283 pJK285 pJK287 pJK289 pJK380 pJK382 pJK410 pJK420 pMW1 pMW47 pMW489 pMW490 pMW526 pRS415 pRS416 pYJ3 pYJ4 pYJ5 pYJ6 pYJ7 pYJ8 pYJ9 pYJ16 pYJ30 pYJ38 pYJ74 pYJ167 pYJ182 pYJ193 pYJ195 pYJ198 pYJ201 pYJ204 pYJ206 pCG60-DBF4N!99 T105A pCG60-DBF4N!93 T105A pET24a-GST-RAD532-164 R70A pGAD-C1-DUN11-160 pGAD-C1-FAR1061-227 pGAD-C1-FHL1253-400 pGAD-C1-FKH141-185 pGAD-C1-MEK11-152 pGAD-C1-XRS21-125 pGAD-C1-FKH21-254 pGAD-C1-PML154-204 pET24a-GST-RAD53483-821 pET24a-GST-RAD53549-730 pYJ380 R605A pET24a-GST-RAD53523-821 pAcPK30-DBF41-704 pAcSG2-HAHIS6-CDC71-507 pRS415-DBF41-704 pRS416-DBF41-704 pRS415-DBF4N!65 LEU2 ARS-CEN URA3 ARS-CEN pCG60-DBF4!67-81 pCG60-DBF4!67-88 pCG60-DBF4!67-93 pCG60-DBF4!67-99 pCG60-DBF4!67-103 pCG60-DBF4!67-107 pCG60-DBF4N!109 pCG60 S84A pCG60 R83E pCG60-DBF4!82-88 pMW489-DBF4!82-88 pCG60 S92A pAcSG2-DBF4!82-88 pMW489-DBF4!76-109 pMW489-DBF4!82-109 pMW489-DBF4!66-109 pMW489-DBF4N!65-!82-88 pGBKT7-DBF41-704 pYJ204-DBF4!82-88 171 This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study Gabrielse et al., 2006 Gabrielse et al., 2006 Gabrielse et al., 2006 Gabrielse et al., 2006 Gabrielse et al., 2006 Sikorski and Hieter, 1989 Sikorski and Hieter, 1989 Chen and Weinreich, 2010 Chen and Weinreich, 2010 Chen and Weinreich, 2010 Chen and Weinreich, 2010 Chen and Weinreich, 2010 Chen and Weinreich, 2010 Chen and Weinreich, 2010 Chen and Weinreich, 2010 Chen and Weinreich, 2010 Miller et al., 2009 Chen and Weinreich, 2010 Chen and Weinreich, 2010 Chen and Weinreich, 2010 This study This study This study Chen and Weinreich, 2010 Miller et al., 2009 Miller et al., 2009 Table 4. (cont’d) pYJ218 pYJ219 pYJ222 pYJ308 pYJ319 pYJ326 pYJ332 pYJ336 pYJ340 pYJ355 pYJ368 pYJ372 pYJ380 pYJ384 pYJ388 pYJ392 pYJ394 pYJ422 pYJ424 pYJ426 pYJ428 pYJ461 pYJ462 pYJ464 pYJ466 pYJ489 pYJ491 pYJ493 pYJ494 pYJ497 pYJ507 pYJ512 pYJ535 pMW489-DBF4!89-109 pMW489-DBF4!100-109 pMW489-DBF4!94-109 pGAD-C1-RAD531-300 pGAD-C1-RAD531-821 pCG60-DBF4!89-93 pCG60-DBF4!100-109 pCG60 T105A pMW489-DBF4!82-88-!100-109 pYJ308 R70A pCG60-DBF466-190 pCG60-DBF466-150 pGAD-C1-RAD53483-821 pYJ319 R70A pYJ319 R605A pCG60 T105E pCG60 T105D pAcSG2-DBF4!100-109 pAcSG2-DBF4!82-88-!100-109 pMW489-DBF4N!65-!100-109 pAcSG2-RAD531-821 pYJ204 R83E pYJ204-DBF4!100-109 R83E pYJ204-DBF4!100-109 pYJ204-DBF4!82-88-!100-109 pCG60 E101K pCG60 R103E pCG60 Q113A pYJ204-DBF4N!81-!100-109 pYJ204-DBF4N!93-!100-109 pCG60 E108D pCG60 T138A pGAD-C1-DBF466-227 172 This study This study This study This study This study Chen and Weinreich, 2010 This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study Table 5. Yeast Strains Stain PJ69-4A W303-1A y57 y59 y205 y1853 y2573 M517 M895 M1261 M1589 M1800 M3581 M3831 M3890 M3905 M3913 M3920 Genotype MATa trp1-901 leu2-3, -112 ura3-52 his3-200 gal4! gal80! LYS2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZ MATa ade2-1, ura3-1 his3-11, -15 trp1-1 leu2-3, -112 can1100 W303 MATa rad53-R70A sml1!::HIS3 W303 MATa rad53-K227A sml1!::HIS3 W303 MATa rad53-R605A sml1!::HIS3 W303 MATa sml1"::URA3 sld3-38A-10his13MYC::kanMX4 W303 MATa dbf4!::TRP1 his3::PDBF4-dbf4 4A::HIS3 sld338A-10his-13MYC::kanMX4 W303 MATa rad53-1 W303 MATa dbf4!::kanMX6 [pMW490; pRS416-DBF4 URA3] W303 MATa dbf4-N!109 W303 MATa rad53-1 dbf4!::kanMX6 [pMW490; pRS416DBF4 URA3] W303 MAT1 dbf4-N!109-kanMX6 W303 MATa rad53!::TRP1 sml1!::HIS3 dbf4!::kanMX6 [pMW490; pRS416-DBF4 URA3] W303 MATa RAD53-3MYC-TRP1 W303 MATa dbf4-N!109-natMX4 W303 MATa dbf4-N!109-natMX4 sld3-38A-10his13MYC::kanMX4 W303 MATa dbf4-N!109-kanMX6 sml1::HIS3 W303 MAT! RAD53-3MYC-TRP1 dbf4-N!109-kanMX6 sml1!::HIS3 173 Source James et al., 1996 Thomas and Rothstein, 1989 Pike et al., 2004 Pike et al., 2004 Pike et al., 2004 Zegerman and Diffley, 2010 Zegerman and Diffley, 2010 Cabrielse at al., 2006 Cabrielse at al., 2006 Cabrielse at al., 2006 Cabrielse at al., 2006 Miller et al., 2009 This study This study This study This study This study This study Table 6. Peptides Peptide name Biotin-Dbf4 (98-113) Biotin-pDbf4 Dbf4 (98-113) pDbf4 (pThr105) pDbf4-V104A pDbf4-E108A pDbf4-E108D pDbf4-L109A Peptide sequence Biotin- KNV EPR VTP KEL LEW Q Biotin- KNV EPR V(pT)P KEL LEW Q KNV EPR VTP KEL LEW Q KNV EPR V(pT)P KEL LEW Q KNV EPR A(pT)P KEL LEW Q KNV EPR V(pT)P KAL LEW Q KNV EPR V(pT)P KDL LEW Q KNV EPR V(pT)P KEA LEW Q Biotin-Rad9 Biotin-pRad9 pSpc72 Biotin- IMS EVE LTQ ELP EVE Biotin- IMS EVE L(pT)Q ELP EVE EEF LSL AQS (pS)PA GSQ LES RD 174 Length Biotin + 17 Biotin + 17 17 17 17 17 17 17 15 15 20 MW 2192.9 2273.2 1966.4 2047.5 2019.8 1989.9 2032.7 2005 1972.28 2052.26 2231.3 ACKNOWLEDGMENTS We thank FuJung Chang (Weinreich Lab) and Feng-Ling Tsai (Schwacha Lab, University of Pittsburgh) for technical help or advice. We also thank John Diffley and Jörg Heierhorst for yeast strains. 175 BIBLIOGRAPHY 176 BIBLIOGRAPHY Bartek J, Lukas C, Lukas J. 2004. Checking on DNA damage in S phase. Nat Rev Mol Cell Biol 5: 792-804. 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EMBO J 20: 3544-3553. 181 CHAPTER 4 FUNCTIONAL CHARACTERIZATION OF THE DBF4 N-TERMINUS BY A GENOME-WIDE SYNTHETIC LETHALITY SCREEN Ying-Chou Chen, Jessica Kenworthy, Charles Boone, and Michael Weinreich This chapter was taken entirely from a manuscript currently in preparation. 182 FUNCTIONAL CHARACTERIZATION OF THE DBF4 N-TERMINUS BY A GENOME-WIDE SYNTHETIC LETHALITY SCREEN ABSTRACT The Dbf4-dependent Cdc7 kinase (DDK) is a conserved two-subunit kinase required for eukaryotic DNA replication. We recently reported that Dbf4 interacts with Cdc5 (yeast Polo-like kinase) and Rad53 (the ortholog of mammalian Chk2 kinase), and proposed that Dbf4 serves a molecular scaffold to assemble a ternary complex (Dbf4-Cdc7-Cdc5-Rad53) that coordinates chromosome segregation and checkpoint signaling pathways in post-replicative cell-cycle regulation. Since dbf4 mutants unable to interact either with Cdc5 or Rad53 exhibit normal cell-cycle progression and grow well under various genotoxic stresses, we suggest that other pathways act in parallel with Dbf4 function. We report here the results of a genome-wide screen for genes that are synthetically lethal or sick in combination with a dbf4 N-terminal deletion, which is defective in binding both Cdc5 and Rad53. This genetic interaction network showed that Dbf4 is involved in multiple surveillance mechanisms that control genome stability (Sgs1, Top3, and Rmi1), DNA replication or damage checkpoint signaling (Rad17, Mec3, Rad24, Rad9, Rad54, Pol32, and Bmh1), and chromosome segregation (Csm1, Ctf18, Ctf8, Dcc1, and the HIR complex). These data not only provide insight into the role of Dbf4 in the convergence of checkpoint signaling and mitotic regulation, but also contribute to a comprehensive understanding of Dbf4 function in cell-cycle regulation. 183 INTRODUCTION In many signaling networks, scaffold proteins are known to recruit pathway components and facilitate the specificity of signal transduction (Burack and Shaw 2000; Ubersax and Ferrell 2007). Although binding partners of a scaffold protein can be determined using biochemical approaches that discover protein-protein interactions, little is known about the biological outcome of the scaffoldmodulated assembly. The signals that transmit though a scaffold protein can be identified by genetic mapping of scaffold mutants, which provides a global view of the functional relationships between genes and pathways (Boone et al. 2007; Dixon et al. 2009). Dbf4 is an essential regulator of the S-phase kinase Cdc7 (also known as Dbf4dependent kinase, DDK), which directly phosphorylates and activates the DNA helicase Mcm2-7 for DNA synthesis (Sclafani 2000; Bell and Dutta 2002). Work in the budding yeast Saccharomyces cerevisiae has shown that an Mcm mutation, mcm5-bob1, bypasses the deletion of the essential genes DBF4 and CDC7, suggesting that Mcm2-7 is the main physiological substrate of DDK (Hardy et al. 1997; Johnston et al. 1999; Sclafani et al. 2002). Structural studies of an archaeal Mcm complex containing an analogous mutation indicate that the genetic suppression is the result of a conformational change that probably mimics the activation of Mcm2-7 helicase (Hoang et al. 2007). Also, DDK phosphorylation has been shown to be required to recruit Cdc45 and the GINS complex to the Mcm2-7 helicase (Owens et al. 1997; Zou and Stillman 2000; 184 Masai et al. 2006; Yabuuchi et al. 2006). Cdc45, Mcm2-7, and GINS form an active replicative helicase that plays a major role in the initiation of DNA synthesis (Weinreich and Stillman 1999; Gambus et al. 2006; Moyer et al. 2006; Sheu and Stillman 2006; Francis et al. 2009). Dbf4 orthologs have been identified in Schizosaccharomyces pombe (dfp1+), Drosophila melanogaster (chiffon), Xenopus laevis (XDBF4), mice (MmDbf4), and humans (HsDbf4/ASK) (Brown and Kelly 1998; Jiang et al. 1999; Kumagai et al. 1999; Landis and Tower 1999; Lepke et al. 1999; Johnston et al. 2000; Furukohri et al. 2003). Multiple sequence alignment of Dbf4 proteins across species revealed three conserved motifs, termed motif-N, -M, and -C (Masai and Arai 2000). It is generally thought that Dbf4 motifs-M and -C bind to and activate Cdc7 kinase (Jones et al. ; Harkins et al. 2009), while the Dbf4 N-terminus (residues 1-296) is dispensable for the essential function of Dbf4 in DNA replication (Duncker et al. 2002; Gabrielse et al. 2006; Miller et al. 2009; Chen and Weinreich 2010). Recently, we characterized two distinct binding motifs within the Dbf4 N-terminus that interact independently with the mitotic Polo-like kinase Cdc5 and the checkpoint kinase Rad53 (Miller et al. 2009; Chen and Weinreich 2010; Chen et al. 2012). Importantly, Cdc5, Rad53 and Cdc7 can form a stable complex with Dbf4 (Chen et al. 2012). CDC5 is the single Polo ortholog in budding yeast and plays multiple essential roles in mitotic and meiotic regulation (Sunkel and Glover 1988; Barr et al. 2004; Archambault and Glover 2009). Genetic evidence suggests that Dbf4 inhibits Cdc5 through a direct 185 interaction to prevent premature exit from mitosis (Miller et al. 2009; Chen and Weinreich 2010). Intriguingly, this Cdc5 inhibition depends on the association between Cdc7 and Dbf4, suggesting that Dbf4 serves as a scaffold for Cdc7 to mediate the Cdc5 inhibition in mitotic exit. When replication forks stall, the active Rad53 kinase binds to the Dbf4 Nterminus in a phosphorylation-dependent manner and phosphorylates the Cterminus of Dbf4, subsequently inhibiting late origin firing (Duch et al. 2010; Lopez-Mosqueda et al. 2010; Zegerman and Diffley 2010; Chen et al. 2012). Although dbf4 mutants that cannot bind either Cdc5 or Rad53 exhibit wild-type growth and normal S-phase progression (Gabrielse et al. 2006; Miller et al. 2009; Chen and Weinreich 2010; Chen et al. 2012), N-terminal deletions of DBF4 are lethal when Rad53 function is compromised, suggesting that Rad53, Cdc5, or both cooperate with DDK to perform an essential cell cycle function (Gabrielse et al. 2006; Chen et al. 2012). However, little is known about how Dbf4 acts in replication fork stability and post-replicative cell-cycle regulation. To identify this, we have performed a genome-wide synthetic lethal screen using the dbf4-N!109 mutant, which is defective in binding both Rad53 and Cdc5. Synthetic genetic interactions are usually identified when a second-site mutation suppresses or enhances the original mutant phenotype. In particular, synthetic lethality occurs when two mutations are separately viable but their combination results in lethality or a reduced fitness that is more severe than that of the 186 individual single mutations. In principle, when two genes show a synthetic lethal interaction, it often reflects the gene products operating in parallel pathways or participating in the same protein complex (Hartman et al. 2001). With its genetic tractability and short generation time, S. cerevisiae has become a powerful model for large-scale mapping of synthetic genetic interactions. A yeast library that collected non-essential deletions has been developed for a Synthetic Genetic Array (SGA) (Giaever et al. 2002; Tong et al. 2004; Huang and Kolodner 2005; Pan et al. 2006b; Boone et al. 2007). We set up a genome-wide SGA screen using the dbf4-N!109 mutant to query nearly 4300 deletion mutants (representing 82% of the genes in budding yeast) for synthetic genetic interactions. The genetic interactions that we uncovered represent a small number of functional categories, including control of genome stability (the Top3- Sgs1-Rmi1 complex), DNA damage or checkpoint signaling (the 9-1-1 complex, Rad54, Rad9, and Bmh1), and chromosome segregation (the CTF and HIR complexes and Csm1). We also described a two-hybrid interaction between Dbf4 and Bmh1 that maps to site overlapping the Rad53 binding site on Dbf4. These results strongly suggest that Dbf4 N-terminus plays a parallel role in the Rad53-mediated checkpoint activation, as well as highlighting that Dbf4 coordinates multiple checkpoint responses through the interactions with various cell-cycle regulators. 187 RESULTS An SGA screen for the dbf4-N!109 mutant Our earlier reports indicated that the Dbf4 N-terminus (residues 1-109) interacts with two essential kinases, Cdc5 and Rad53 (Gabrielse et al. 2006; Miller et al. 2009; Chen and Weinreich 2010; Chen et al. 2012). The dbf4-N!109 allele not only suppresses the temperature sensitivity of the cdc5-1 mutant, but also shows a synthetic lethal interaction with rad53-1, a hypomorphic mutant of the RAD53. Despite advanced studies in molecular interactions, the biological function of the Dbf4-associated complexes is poorly understood, because N-terminal deletions of dbf4 do not show any significant phenotype, even with various genotoxic treatments. This may be due to a redundant pathway that acts in parallel with Dbf4 function. Thus, we performed an SGA analysis using the dbf4-N!109 mutant, which abolishes the interaction with Cdc5 and Rad53, as the query strain to probe the specific function of the Dbf4 N-terminus on a genome-wide scale. We first constructed the dbf4-N!109 mutant and integrated a nourseothricin resistance marker (natMX4) in its 3’ UTR. This query mutant was crossed with the 4,293 viable yeast deletion mutants on the SGA. The yeast mating-type alpha (MAT!) strain carrying dbf4-N!109::natMX4 was systematically crossed onto the SGA, on which all the deletion mutants were in the mating-type a (MATa) (Tong et al. 2004). Strains showing resistance to both nourseothricin and geneticin were selected as diploid strains. The resulting heterozygous diploids were transferred to a medium with reduced carbon and nitrogen to induce 188 sporulation and the formation of haploid spore progeny. Spores were further transferred to a synthetic medium that specifically selects for germination of MATa through the engineered mating-type reporter (MFA1pr-HIS3) (Pan et al. 2004). Two recessive markers, can1! and lyp1!, which confer drug resistance to canavanine and thialysine, were also used as haploid-selectable markers (Baryshnikova et al. 2010). The MATa meiotic progeny were then transferred onto medium contained both nourseothricin and geneticin for the selection of double mutants. A synthetic lethal or synthetic sick interaction was identified when the colony size of double-mutant progeny was smaller than that of wildtype controls. For the purpose of computer-based scoring, the adjusted calibrated p-values were calculated by comparing the measurements between the mutants and wild-type controls (Tong et al. 2004). Following normalization, statistical significance was indicated by the values of t-statistics. Table 7 lists the validated synthetic lethal and synthetic sick interactions with dbf4-N!109 from our SGA screen. The genetic interaction profile of dbf4-N!109 partially overlapped with the SGA profiles of the dbf4-1 and cdc7-1 hypomorphic mutants (Tong et al. 2004) (Table 8 and data not shown). Genes involved in maintaining chromosome stability (TOP3, SGS1, and YLR235C), in the CTF complex (CTF18, CTF8, and DCC1), in the 9-1-1 complex and checkpoint signaling (RAD17, MEC3, and BMH1), and in chromatin structure and chromosome segregation (HIR1, HIR3, and HPC2) were found in the three screens. We also identified genes that specifically interact with the dbf4-N!109 189 allele, including RMI1, POL32, RAD54, HIR2, ASF1, CSM1, BUB3, and a handful of transcriptional regulators (LSM7, CDC73, SRB2, MED1, and CKB2). Numerous candidates had sequence similarity to human genes or analogous functions, so we focused further analysis and interpretation on those genes. 190 Table 7. Synthetic lethality or sickness with dbf4-N!109 Rank 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 ORF YKL139W YLR235C YPL024W YNL147W YGR270W YMR237W YLR418C YBR215W YOR368W YMR190C YOR026W YPL213W YPL178W YLR338W YPL079W YOR039W YCR077C YHR191C YHR041C YOR297C YMR205C YCR086W YJR043C YPR070W YER173W Gene CTK1 TOP3* RMI1 LSM7 YTA7 BCH1 CDC73 HPC2 RAD17 SGS1 BUB3 LEA1 CBC2 OPI9 RPL21B CKB2 PAT1 CTF8 SRB2 TIM18 PFK2 CSM1 POL32 MED1 RAD24 Gene Function Transcriptional regulator Top3-Sgs1-Rmi1 complex, recombination, genome stability Top3-Sgs1-Rmi1 complex, recombination, genome stability Transcriptional regulator Transcriptional regulator Intracellular trafficking Transcriptional regulator HIR complex, heterochromatin formation and kinetochore assembly 9-1-1 clamp complex, DNA damage checkpoint Top3-Sgs1-Rmi1 complex, recombination, genome stability Spindle checkpoint Transcriptional regulator Transcriptional regulator Cytoskeletal organization Biosynthesis Transcriptional regulator Transcriptional regulator CTF complex, sister chromatid cohesion Transcriptional regulator Intracellular trafficking Glycolysis Chromosome segregation DNA replication Transcriptional regulator 9-1-1 clamp complex, DNA damage checkpoint 191 Table 7. (cont’d) 34 YMR078C CTF18 CTF complex, sister chromatid cohesion 36 YLR234W TOP3 Top3-Sgs1-Rmi1 complex, recombination, genome stability 39 YOR038C HIR2 HIR complex, heterochromatin formation and kinetochore assembly 43 YGL163C RAD54 DNA double-strand break repair 45 YJL115W ASF1 HIR complex, heterochromatin formation and kinetochore assembly 47 YBL008W HIR1 HIR complex, heterochromatin formation and kinetochore assembly 48 YJR140C HIR3 HIR complex, heterochromatin formation and kinetochore assembly 49 YCL016C DCC1 CTF complex, sister chromatid cohesion 70 YDR363W ESC2 Chromatin silencing 93 YER177W BMH1 14-3-3 homolog * open reading frame partially overlaps the gene TOP3 192 Table 8. Summary of common hits in the SGA screens dbf4-N!109 YLR235C RAD17 CTF8 RAD24 TOP3 RAD54 HIR1 HIR3 DCC1 ESC2 RMI1 HPC2 BMH1 SGS1 CKB2 CTF18 dbf4-1* YLR235C RAD17 CTF8 RAD24 TOP3 RAD54 HIR1 HIR3 DCC1 ESC2 RMI1 HPC2 BMH1 CHK1 IRA2 STI1 RAD9 RTT107 TIF1 MEC3 * Tong et al., Science (2004) cdc7-1* YLR235C RAD17 CTF8 RAD24 TOP3 RAD54 HIR1 HIR3 DCC1 ESC2 SGS1 CKB2 CTF18 CHK1 IRA2 STI1 RAD9 RTT107 TIF1 MEC3 193 Gene Function Top3-Sgs1-Rmi1 complex 9-1-1 clamp complex CTF complex 9-1-1 clamp complex Top3-Sgs1-Rmi1 complex DNA repair HIR complex HIR complex CTF complex Chromatin silencing Top3-Sgs1-Rmi1 complex HIR complex 14-3-3 homolog Top3-Sgs1-Rmi1 complex Transcriptional regulator CTF complex DNA repair Signal transduction Protein folding DNA repair DNA repair Glycolysis 9-1-1 clamp complex Dbf4 has strong synthetic interactions with the Top3-Sgs1-Rmi1 complex Three of our top ten hits mapped to the Top3-Sgs1-Rmi1 complex, which is related to maintenance of genomic integrity (summarized in Table 7). Top3 is a type IA topoisomerase that resolves the catenation of chromosomes in replication forks (Mankouri and Hickson 2007; Suski and Marians 2008; Cejka et al. 2012). Deletion of the TOP3 gene results in elevated levels of recombination at repetitive sequences, which subsequently leads to abnormal chromosomal translocation and rearrangement, an increased rate of sister chromatid exchanges and chromosome loss, and slow growth (Wallis et al. 1989; Myung et al. 2001). The DNA helicase Sgs1, one of the RecQ family of DNA helicases, was identified as a slow growth suppressor of the top3-null mutant (Gangloff et al. 1994). The physical interaction between Top3 and Sgs1 is evolutionarily conserved from yeast to humans (Harmon et al. 1999; Bennett et al. 2000; Wu et al. 2000; Harmon et al. 2003). Three of five RecQ homologs in the human genome are associated with rare genetic diseases: Bloom (BLM), Werner (WRN), and Rothmund-Thomson (RecQ4) syndromes, which were characterized by genomic instability, predisposition to cancer, and premature aging. The budding yeast gene SGS1 is most homologous to human BLM (Chu and Hickson 2009). Two independent genetic screens identified Rmi1 as a third member of the Top3Sgs1 complex (Chang et al. 2005; Mullen et al. 2005). Rmi1 physically associates with Top3 and Sgs1, and is required for the Top3-Sgs1 function of ressolving Holliday junctions to complete recombination (Cejka et al. 2010a; 194 Cejka et al. 2010b; Hickson and Mankouri 2011; Cejka et al. 2012). To validate the genetic interaction, which was performed in yeast strain BY4741 (a derivative in the S288C background), we integrated dbf4-N!109 and rmi1! into a commonly used yeast strain, W303, to retest the synthetic effects. W303 cells harboring a deletion of RMI1 grew slowly, whereas dbf4-N!109 mutants underwent normal cell cycle progression. Tetrad analysis by crossing rmi1! to dbf4-N!109 showed that the double mutants were synthetically lethal (Figure 26A and Table 9). Similarly, we placed the sgs1! allele in the W303 background and confirmed that dbf4-N!109 was synthetically sick with sgs1! (Figure 26B). YLR235C is a dubious open reading frame, which overlaps the C-terminal portion of the TOP3 gene, likely resulting in a top3 hypomorphic mutant. YLR235C! exhibits a strong synthetic effect with dbf4-N!109 in the S288C background. Attempts at introducing a YLR235C null mutation into W303 caused severe defects in growth that were similar to those of the top3! mutant. Therefore, we used a W303 top3 null mutation (top3-2) in which the growth defects are suppressed by the sgs1-3 allele (Lu et al. 1996). The top3-2 sgs1-3 strain was crossed to the dbf4-N!109 strain for tetrad analysis. The phenotypes of the resulting colonies indicated that the top3-sgs1-dbf4 triple mutants were synthetic lethal or sick, even though the growth of the top3-sgs1 double mutants resembled that of the wild-type strain (Figure 26C). Taken together, we found strong genetic interactions with dbf4-N!109 and of all components of the Top3Sgs1-Rmi1 complex in both the S288C and W303 backgrounds. 195 To identify signaling pathways that might be regulated by these genetic interactions, we compared the dbf4-N!109 data with previous SGA profiles that probed individually for top3!, sgs1!, or rmi1!. A small group of gene functions were significantly enriched (data not shown), particularly those directly involved in DNA metabolism and genomic maintenance (the Ctf18-Ctf8-Dcc1 complex, Asf1, Pol32, Csm1, Pat1, Rad24, Rad53, and Rad54). These results indicate that there is an extensive crosstalk in which Dbf4 and the Top3-Sgs1-Rmi1 complex share redundant mechanisms to control genome integrity. 196 Figure 26. The Dbf4 N-terminus genetically interacts with the Top3-Sgs1-Rmi1 complex dbf4-N!109 is synthetically lethal or synthetically sick with rmi1!, sgs1!, and top3-2 sgs1-3 in the W303 background. Representative tetrads from diploid strains of genotype (A) DBF4/dbf4-N!109 RMI1/rmi1!, (B) DBF4/dbf4-N!109 SGS1/sgs1!, and (C) DBF4/dbf4-N!109 TOP3/top3-2 SGS1/sgs1-3 were sporulated and dissected onto YPD plates. Recombinant genotypes are indicated. Detailed GO annotations are summarized in Table 9. 197 Figure 26. (cont’d) A B C top3-2 sgs1-3 198 Table 9. Synthetic genetic interaction between dbf4-N!109 and the BLM complex in W303 TOP3 YLR234W Synthetic lethal/sick Topoisomerase DNA Topoisomerase III, conserved protein that functions in a complex with Sgs1 and Rmi1 to relax single-stranded negatively-supercoiled DNA, involved in telomere stability and regulation of mitotic recombination. BLM complex (Top3-Sgs1-Rmi1) SGS1 RMI1 YMR190C YPL024W Synthetic sick Synthetic lethal RecQ Mediated genome Slow Growth Suppressor Instability Nucleolar DNA helicase Subunit of the Top3of the RecQ family, Sgs1 complex, involved in genome stimulates superhelical integrity maintenance; relaxing and ssDNA regulates chromosome binding activities of synapsis and meiotic Top3, involved in joint crossover response to DNA formation, similar to damage, null mutants human BLM and WRN display increased rates proteins implicated in of recombination and Bloom and Werner delayed S phase. syndromes. 199 YLR235C N/D Dubious open reading frame unlikely to encode a protein, based on available experimental and comparative sequence data, partially overlaps the verified gene TOP3. Genetic and functional interactions between Dbf4 and the CTF complex In the 50 highest scoring candidates from our SGA screen, we found CTF18, CTF8, and DCC1, which represent all of the non-essential components in the CTF (chromosome transmission fidelity) complex (Hanna et al. 2001; Mayer et al. 2001; Naiki et al. 2001). The CTF complex is an alternative RFC (replication factor C) and shares four common subunits (Rfc2, Rfc3, Rfc4, and Rfc5) with Rfc1-5 (the canonical RFC complex). Rfc1 is replaced by Rad24, Ctf18-Ctf8Dcc1 or Elg1 to give three alternative RFC-like complexes involved in various DNA metabolism (Green et al. 2000; Majka and Burgers 2004; Aroya and Kupiec 2005; Kim et al. 2005; Shiomi et al. 2007). The Ctf18-Ctf8-Dcc1-RFC complex (termed Ctf18-RFC) is required for proper cohesion of sister chromatids in DNA replication, and mutations in these genes cause chromosome mis-segregation (Wang et al. 2000; Edwards et al. 2003; Lengronne et al. 2006). Recent studies indicate that Ctf18-RFC also participates in a Rad53 checkpoint that guards fork stability during replication stress (Pan et al. 2006a; Crabbe et al. 2010). The ctf18, ctf8 and dcc1 null alleles were introduced separately into the W303 strain to validate their synthetic genetic interactions with dbf4-N!109. The ctf dbf4 double mutants had slower growth phenotypes in tetrad analyses (data not shown) and they exhibited striking temperature-sensitive phenotypes (Figure 27A). The double mutants also showed increased sensitivity to hydroxyurea (HU), methyl methanesulfonate (MMS) or benomyl (Figure 27B-D). The synergistic combination of genetic alternation and chemical treatment suggests a complex 200 genetic network between Dbf4 and the CTF complex. Comparison between the SGA results of dbf4-N!109 and CTF null mutants identified a subgroup of genes that are closely related to checkpoint activation (TOP3, SGS1, RAD17, RAD24, RAD53, RAD54 and POL32) and the regulation of chromosome dynamics (ASF1, CSM1, and BUB3) (data not shown). Overall, these results suggest that Dbf4 is involved in an additional level of control for achieving the coordination between checkpoint signal transduction and chromosome segregation. The genetic and functional interactions between Dbf4 and the CTF complex are likely due to a combined defect in Rad53-dependent checkpoint signaling during stresses. It has been previously shown that Dbf4 and Cdc7 function as key regulators for homologous chromosome segregation in meiosis I (Valentin et al. 2006; Matos et al. 2008; Wan et al. 2008; Katis et al. 2010; Lo et al. 2012). DDK collaborates with the Cdc5 kinase to establish the monopolin complex (Mam1-Lrs4-Csm1), which allows kinetochores to specifically attach to spindles from the same spindle pole body (equivalent to the centrosome in higher eukaryotes) (Clyne et al. 2003; Lee and Amon 2003; Marston 2009). Given that dbf4-N!109 is synthetically sick with csm1! in both S288C and W303 (Table 7 and Figure 28A), this suggests that the Dbf4-Cdc5 interaction and Csm1 also work together to accomplish successful chromosome segregation. Similarly, proper control of chromosome segregation also depends on the centromeric cohesion that may be promoted by the Ctf18-RFC complex. 201 Table 10. Synthetic genetic interaction between dbf4-N!109 and the CTF complex in W303 CTF8 YHR191C temperature sensitive Chromosome Transmission Fidelity Subunit of a complex with Ctf18 that shares some subunits with Replication Factor C and is required for sisterchromatid. CTF18 YMR078C temperature sensitive Chromosome Transmission Fidelity Subunit of a complex with Ctf8 that shares some subunits with Replication Factor C and is required for sisterchromatid cohesion, have overlapping functions with Rad24p in the DNA damage replication checkpoint. 202 DCC1 YPL194W temperature sensitive DNA Damage Checkpoint DNA damage checkpoint protein, part of a PCNAlike complex required for DNA damage response. Figure 27. dbf4 ctf double mutants exhibit synthetic defects in growth upon environmental stresses Serial dilution of log-phase cells of indicated genotypes were spotted on YPD medium (A) at various temperatures and on the YPD medium that contained (B) HU, (C) MMS, or (D) benomyl. A 25ºC 30ºC WT B 0.1 M HU WT 203 37ºC Figure 27. (cont’d) C D 0.03% 0.04% MMS WT 15 !g/ml benomyl WT 204 Figure 28. The Dbf4 N-terminus genetically interacts with Csm1, Pol32, and Rad54 dbf4-N!109 is synthetically sick with csm1!, pol32!, and rad54! in the W303 background. Representative tetrads from diploid strains of genotype (A) DBF4/dbf4-N!109 CSM1/csm1!, (B) DBF4/dbf4-N!109 POL32/pol32!, and (C) DBF4/dbf4-N!109 RAD54/rad54 were sporulated and dissected onto YPD plates. Recombinant genotypes are indicated. Detailed GO annotations are summarized in Table 11. 205 Figure 28. (cont’d) A B C 206 Table 11. Validation of the dbf4-N!109 SGA results in W303 CSM1 YCR086W Synthetic sick Chromosome Segregation in Meiosis Nucleolar protein that forms a complex with Lrs4 and then Mam1 at kinetochores during meiosis I to mediate accurate homolog segregation, required for condensin recruitment to the replication fork barrier site and rDNA repeat segregation. DNA metabolism POL32 YJR043C Synthetic sick RAD54 YGL163C Synthetic sick POLymerase RADiation sensitive Third subunit of DNA polymerase delta, involved in chromosomal DNA replication, required for error-prone DNA synthesis in the presence of DNA damage and processivity. DNA-dependent ATPase, stimulates strand exchange by modifying the topology of double-stranded DNA, involved in the recombinational repair of DNA double-strand breaks. 207 DDK participates in DNA damage checkpoint response The fission yeast Dbf4-dependent kinase (Hsk1) has been recently shown to phosphorylate the PCNA-like 9-1-1 clamp (Ddc1-Rad17-Mec3; named Rad9Rad1-Hus1 in S. pombe and humans) in response to DNA damage (Furuya et al. 2010). It is thought that the heterotrimeric complex of 9-1-1, with its clamp loader Rad24-RFC, acts as a DNA damage sensor, which binds Dbp11 (the homolog of TopBP1) to recruit the checkpoint kinase Mec1 (functional homolog of ATR in mammals) during checkpoint activation (Majka et al. 2006; Labib and De Piccoli 2011). The subsequent signal transduction relies on Rad9 (S. cerevisiae) as an adaptor to activate the checkpoint effector kinases Rad53 and Chk1, which are integral transducers of cellular responses to genotoxic stress (Branzei and Foiani 2009). We have previously shown that the N-terminal deletions of dbf4 mutants are synthetically lethal with mec1-1, rad53-1, rad53! sml1!, or chk1! (Duncker et al. 2002; Gabrielse et al. 2006; Chen et al. 2012). Furthermore, mec3!, chk1!, and rad9! have separately been shown to be synthetically sick with dbf4-1 or cdc7-1 (Tong et al. 2004). Here, we found that rad24! and rad17! were synthetically sick with dbf4-N!109 in the S288C background (summarized in Table 7). These data confirmed the genetic interaction of DBF4 with the DNA damage checkpoint pathway. However, components of the 9-1-1 clamp or Rad24 did not exhibit synthetic growth phenotype with dbf4-N!109 in W303 strains. 208 Dbf4 genetically interacts with the HIR complex and RNA modulators In budding yeast, the HIR (histone regulatory) and CAF-1 (chromatin assembly factor 1) complexes are known as histone chaperones and are involved in nucleosome deposition and chromatin-mediated transcriptional silencing (Osley and Lycan 1987; Smith and Stillman 1989; Xu et al. 1992; Kaufman et al. 1997; Dimova et al. 1999). It is generally thought that CAF-1 participates in the replication-coupled nucleosome assembly via a direct interaction with PCNA (proliferating cell nuclear antigen), while the HIR complex mainly regulates the histone dynamics outside of S phase (Green et al. 2005). HIR1, HIR2, HIR3, and HPC2 encode the subunits of the HIR complex; CAC1, CAC2, and CAC3/MSI1 encode the subunits of the CAF-1 complex. The growth of S. cerevisiae cells is not affected when either HIR or CAC genes are mutated, but hir! cac! doublemutants exhibit slow growth, suggesting that two complexes functionally overlap (Kaufman et al. 1998; Qian et al. 1998). Both complexes interact with a conserved H3/H4-binding protein Asf1 (anti-silencing function), which is found in a complex with the checkpoint kinase Rad53 (Emili et al. 2001; Hu et al. 2001; Sharp et al. 2005; Jiao et al. 2012). A compelling model is that a Rad53mediated checkpoint response prevents the deposition of newly synthesized histones in the cells upon DNA damage and replication stress and thus contributes to genome integrity (Hu et al. 2001; Singh et al. 2009). In our SGA screen, hpc2!, asf1!, hir1!, hir2!, and hir3! were among the top 50 candidates that were synthetically sick with dbf4-N!109 (summarized in Table 7). 209 In particular, !hpc2, hir1! and hir3! were found earlier to be synthetically sick with dbf4-1 or cdc7-1. None of the CAF-1 components was identified in our SGA screen, indicating that Dbf4 is specifically relevant to HIR-associated histone homeostasis, and separable from the role of CAF-1 within S phase. In addition to acting as histone chaperones, the HIR-Asf1 complex is also involved in gene silencing, the repression of histone genes, nucleosome disassembly, and aspects of transcriptional regulation (Sharp et al. 2002; Prochasson et al. 2005; Amin et al. 2012; Eriksson et al. 2012; Zunder and Rine 2012). Whether and how Dbf4 participates in these mechanisms has not been investigated yet, but it is intriguing to find that one third of the top hits in the dbf4N!109 SGA screen are known to be involved in transcriptional regulation. Although it is likely that these genetic interactions come from indirect effects, it is noteworthy that most synthetic effects with RNA metabolism genes were confirmed in the W303 background (summarized in Table 12). In particular, the deletion of the LSM7, CDC73, SBR2, CKB2, and MED1 genes showed significant synthetic effects with dbf4-N!109 (Figure 29). Dbf4 physically and genetically interacts with the yeast 14-3-3 proteins We recently showed that the checkpoint kinase Rad53 directly binds to the Dbf4 N-terminus using both FHA1 and FHA2 domains (Chen et al. 2012). Rad53 also interacts with Bmh1 and Bmh2, which are homologs of the 14-3-3 protein family and can form homo- or heterodimers to regulate diverse biological processes, 210 including signal transduction in G1/S and G2/M checkpoints, DNA replication, genome stability, apoptosis, cytoskeleton organization, and malignant transformation (Lottersberger et al. 2003; Usui and Petrini 2007; Grandin and Charbonneau 2008; Freeman and Morrison 2012; Gardino and Yaffe 2012). At least seven isoforms of 14-3-3 are present in humans, but BMH1 and BMH2 are the only two 14-3-3 genes in budding yeast. We found that Bmh1 and Bmh2 associate with Dbf4 in yeast two-hybrid assays (Figure 30). Surprisingly, both yeast 14-3-3 proteins bind to Dbf4 by recognizing a sequence overlapping the Rad53 binding site, suggesting that Rad53 recruits Bmh1 and Bmh2 to associate with Dbf4. Interestingly, bmh1! is one of the candidates showing a synthetic sick interaction with dbf4-N!109 in the SGA screen, but bmh2! is not. We observed similar results in the W303 background by tetrad analyses. As shown in Figure 31, the bmh1! dbf4-N!109 double mutant is synthetically sick and synergistically sensitive to benomyl treatment, whereas bmh2! dbf4-N!109 double mutants had no such growth phenotype (data not shown). Since dbf4-N!109 is synthetically lethal with rad53-1, we tested whether bmh1! had synthetic effects with rad53-1. As expected, bmh1! rad53-1 double mutants had more severe growth defects than did the bmh1! dbf4-N!109 mutants (Figure 31B). These genetic data suggest that the functional link between Dbf4 and Bmh1 relies on the participation of Rad53, which probably promotes the interaction between Dbf4 and Bmh1. 211 Figure 29. The Dbf4 N-terminus is involved in transcriptional regulation dbf4-N!109 displays synthetic effects with lsm7!, cdc73!, srb2!, ckb2!, and med1! in the W303 background. Representative tetrads from diploid strains of genotype (A) DBF4/dbf4-N!109 LSM7/lsm7!, (B) DBF4/dbf4-N!109 CDC73/cdc73!, (C) DBF4/dbf4-N!109 SRB2/srb2!, (D) DBF4/dbf4-N!109 CKB2/ckb2!, and (E) DBF4/dbf4-N!109 MED1/med1 were sporulated and dissected onto YPD plates. Recombinant genotypes are indicated. Detailed GO annotations are summarized in Table 12. (F) Serial dilution of log-phase cells of indicated genotypes were spotted on YPD medium at various temperatures. A B 212 Figure 29. (cont’d) C D F 25ºC E 30ºC WT 213 37ºC Table 12. Synthetic genetic interaction between dbf4-N!109 and transcriptional regulators RNA metabolism (transcriptional regulation) LSM7 CDC73 SRB2 YNL147W YLR418C YHR041C Synthetic sick Synthetic sick Synthetic sick Suppressor of RNA Like SM Cell Division Cycle polymerase B Lsm (Like Sm) protein, Component of the Paf1 Subunit of the RNA part of heteroheptameric complex, binds to and polymerase II mediator complexes (Lsm2-7), modulates the activity of complex, associates with involved in mRNA decay RNA polymerases I and core polymerase and processing of tRNA, II, required for gene subunits to form the snoRNA, and rRNA. expression, histone RNA polymerase II modification, and holoenzyme, involved in telomere maintenance. telomere maintenance. 214 Table 12. (cont’d) RNA metabolism (transcriptional regulation) CKB2 MED1 YOR039W YPR070W Temperature sensitive Synthetic lethal Casein Kinase Beta' MEDiator complex subunit Beta' regulatory subunit Subunit of the RNA of casein kinase 2 polymerase II mediator (CK2), a Ser/Thr protein complex, associates with kinase with roles in cell core polymerase growth and proliferation, subunits to form the CK2, comprised of RNA polymerase II CKA1, CKA2, CKB1 and holoenzyme. CKB2, has many substrates including transcription factors and all RNA polymerase. 215 Figure 30. Mapping the interaction between Dbf4 and yeast 14-3-3 protein (A-B) N-terminal Dbf4 deletion or point mutants were tested for a two-hybrid interaction with the Bmh1 and Bmh2, separately. 10-fold serial dilutions of saturated cultures were spotted onto SCM-Trp-Leu plates to visualize total cells and Scm/-Trp-Leu-His + 2 mM 3AT plates, to score the two-hybrid interaction. (C) Schematic of the features in Dbf4 N-terminus are shown, including motifs N, M and C and Cdc5 (Polo) and Rad53 binding sites, along with a summary of the two-hybrid data. 216 Figure 30. (cont’d) A Dbf4 Bait Total cells Y2H interaction Prey Vector Bmh1 66-227aa 72-227aa 77-227aa 82-227aa 88-227aa 94-227aa 100-227aa Bmh1 104-227aa 109-227aa 66-109aa 66-150aa Bmh1 Dbf4 66-227aa 66-190aa FF165,166AA W202A W202E Scm/-Trp-Leu 217 Scm/-Trp-Leu-His +2mM 3AT Figure 30. (cont’d) B Dbf4 Bait Total cells Y2H interaction Prey Vector Bmh2 66-227aa 72-227aa 77-227aa 82-227aa 88-227aa 94-227aa 100-227aa Bmh2 104-227aa 109-227aa 66-109aa 66-150aa Bmh2 Dbf4 66-227aa 66-190aa FF165,166AA W202A W202E Scm/-Trp-Leu 218 Scm/-Trp-Leu-His +2mM 3AT Figure 30. (cont’d) C 1 135 179 N 260 309 M 83 704aa C Dbf4 Rad53 binding Polo 1 656 115 88 100 221 227 66 Bmh1 Rad53 Bmh2 Interaction Interaction Interaction +++ +++ +++ 82 ++ 66 66 66 66 66 FF165, 166AA W202A 66 66 227 219 - - - - - - - - - - - - +++ 227 - - 190 - +++ - - 150 - - 109 +++ - 110 ++ - 104 +++ - 100 ++ + 94 +++ +++ - - - Figure 31. The Dbf4 N-terminus genetically interacts with Bmh1 bmh1 is synthetically sick with dbf4-N!109 and synthetically lethal with rad53-1 in the W303 background. Representative tetrads from diploid strains of genotype (A) DBF4/dbf4-N!109 BMH1/bmh1! and (B) RAD53/rad53-1 BMH1/bmh1! were sporulated and dissected onto YPD plates. Recombinant genotypes are indicated. (C) Serial dilution of log-phase cells of indicated genotypes were spotted on the YPD medium that contained benomyl. A B rad53-1 220 Figure 31. (cont’d) C YPD 15 !g/ml WT 221 22.5 !g/ml 30 !g/ml benomyl DISCUSSION Functional characterization of the Dbf4 N-terminus Dbf4 is well known as a regulatory subunit of the Cdc7 kinase (Johnston et al. 2000; Sclafani 2000). Motif-M and motif-C of Dbf4 are required for the essential function of Cdc7 in DNA replication, but the Dbf4 motif-N is dispensable for yeast viability (Masai and Arai 2000; Gabrielse et al. 2006; Harkins et al. 2009; Jones et al. 2010). In recent years, it has been thought that the Dbf4 N-terminus has separate roles in post-replicative cell-cycle regulation. A series of biochemical and genetic studies identified various binding partners in the Dbf4 N-terminus, including Orc2 and Orc3 (origin recognition complex) (Duncker et al. 2002), Cdc5 (Polo-like kinase) (Miller et al. 2009; Chen and Weinreich 2010), and Rad53 (checkpoint kinase) (Duncker et al. 2002; Chen et al. 2012; Matthews et al. 2012). Among these, the molecular basis of the Dbf4-Cdc5 and Dbf4-Rad53 interactions was extensively studied, but the biological relevance of these physical interactions is not completely understood. Cdc5, Rad53, and Cdc7 simultaneously complex with Dbf4 (Chen et al. 2012), and the ternary complex is likely involved in different surveillance mechanisms in DNA replication checkpoint, G2/M checkpoint adaptation, and spindle position checkpoint. We proposed that Dbf4 serves as a molecular scaffold and that the rewiring of different checkpoint signal transmissions depends on the dynamic complex formation of Dbf4. Since the N-terminal first 109 residues of Dbf4 are required for the Cdc5 and Rad53 interactions (Miller et al. 2009; Chen and Weinreich 2010), a synthetic 222 lethal screen using dbf4-N!109 as a bait was proposed to uncover genes involved in Rad53 or Cdc5 signaling. In fact, the dbf4-N!109 SGA analysis identified a group of genes that control genome integrity, chromosome segregation, and DNA damage response, in which Cdc5 or Rad53 are known to play important roles. These data suggest that the role of Dbf4 N-terminus is complemented by either Cdc5 or Rad53-related pathways, and cells can tolerate the dbf4-N!109 mutation when Cdc5 or Rad53 function is unperturbed. Such an observation is consistent with recent findings that dbf4-N!109 is synthetically lethal with rad53-1 and that the cdc5-1 temperature-sensitive mutant loses viability by introducing a dbf4 N-terminal deletion (Gabrielse et al. 2006; Miller et al. 2009; Chen and Weinreich 2010; Chen et al. 2012). Earlier SGA screens with dbf4-1 and cdc7-1 revealed that DDK genetically interacts with the Top3-Sgs1-Rmi1 complex (Tong et al. 2004), suggesting that DDK is involved in preserving the fidelity of genome inheritance. Unlike the dbf41 and cdc7-1 mutants, cells harboring the dbf4-N!109 allele show no effect in DNA synthesis (Gabrielse et al. 2006). Our present work showed that the dbf4N!109 mutant displays reduced fitness in combination with any mutant in the Top3-Sgs1-Rmi1 complex, strongly suggesting that these synthetic genetic interactions are not due to a defect in DNA replication. Because the Dbf4 1-109 residues are not required for the essential function of Cdc7 in S phase, these results also imply that, in addition to the initiation of DNA synthesis, the Cdc7 kinase has a distinct function linked to the Dbf4 N-terminus. This idea is 223 consistent with our recent finding that Cdc7 is crucial for Dbf4-regulated Cdc5 inhibition during mitotic exit (Miller et al. 2009; Chen and Weinreich 2010). With dbf4-N!109 as a query in the SGA screen, we isolated multiple genes in the 9-1-1, CTF, and HIR complexes. These interacting genes not only reflected novel roles of Dbf4 in chromatin dynamics, but also provided insights into the molecular mechanism. To follow identification of each potential candidate, we validated the SGA results in the W303 background. Twenty of the 34 top-scoring hits showed synthetic effects in W303. rmi1!, sgs1!, and top3! consistently exhibited strong phenotypes with dbf4-N!109. Though the ctf dbf4 double-mutants had mild growth defects under normal growth conditions, they showed increased sensitivity to various genotoxic stresses. It is striking that none of genes in the HIR-Asf1 or 9-1-1 complexes showed synthetic effects with dbf4-N!109 in the W303 background, even though many of them were already identified in previous SGA studies by using the dbf4-1 and cdc7-1 alleles. Further studies on the divergence between different genetic backgrounds are clearly warranted. It’s all about Rad53 activation The genome-wide synthetic lethal screen enables us to take an unbiased approach to studying the biological significance of the dbf4-N!109 allele. Many candidate genes and pathways are involved in the mechanism of Rad53 activation, suggesting that Rad53 is the central node in the genetic and biochemical networks of DBF4. 224 DNA damage response and checkpoint signaling The Top3-Sgs1-Rmi1 and 9-1-1 complexes play important roles in recognizing and processing DNA breaks (Harrison and Haber 2006). In S. cerevisiae, DNA double-strand break (DSB) repair is initiated by end resection. The conserved Mre11-Rad50-Xrs2 (MRX) complex, together with Sae2, recruits the Dna2 nuclease, Exo1 exonuclease, and Top3-Sgs1-Rmi1 helicase complexes to the break sites and removes oligonucleotides from the 5! strand. The resulting ssDNA 3! overhang is then coated by RPA (replication protein A). This ssDNARPA intermediate interacts with the 9-1-1 complex, leading to Tel1/Mec1 (the ATM/ATR kinase homolog in mammals) recruitment and subsequent activation of Rad53 checkpoint kinase. Therefore, a loss of function of the Top3-Sgs1-Rmi1 or 9-1-1 complex can limit Rad53 activation. We previously found that checkpointcompromised rad53 mutants (rad53-1, rad53-11, and rad53! sml1!) were synthetically lethal with dbf4-N!109 (Gabrielse et al. 2006; Chen et al. 2012). Similarly, deleting genes in the Top3-Sgs1-Rmi1 or 9-1-1 complexes caused synthetic lethal or sick phenotypes with dbf4-N!109 in the SGA screen, suggesting that the synthetic genetic interaction may be mediated by a combined defect in Rad53 activation. Furthermore, mec1-1 (the principal kinase for Rad53 activation) was synthetically lethal with dbf4 N-terminal deletions (Gabrielse et al. 2006), and chk1! (partially redundant with Rad53 function) and rad9! (an adaptor for Rad53 activation in response to DNA damage) were synthetically sick with dbf4-1 or cdc7-1 (Tong et al. 2004), indicating that these synthetic genetic 225 interactions reflect complex and partially overlapped mechanisms in the Rad53dependent checkpoint signaling. In support of this notion, previous studies found that deletion of CTF18, CTF8 or DCC1, which were synthetically sick with dbf4N!109 in the SGA screen, caused defects in the Rad53 activation in response to folk stalling (Pan et al. 2006a; Crabbe et al. 2010). Accordingly, the Rad53 kinase responds to genotoxic stress by inducing transcription of genes that modulate cell cycle progression (Bastos de Oliveira et al. 2012; Travesa et al. 2012). The synthetic effects between dbf4-N!109 and several transcriptional regulators may be analogous to a scenario that is conferred by the dbf4-N!109 rad53 double mutants; nonetheless, we do not rule out the possibility that Dbf4 coordinates transcriptional responses to replication stresses or that the synthetic interactions with dbf4-N!109 are indirect. The yeast 14-3-3 proteins, Bmh1 and Bmh2, contribute to a robust activation of checkpoints upon DNA damage and replication stress, as well as two distinct spindle checkpoints (Lottersberger et al. 2003; Usui and Petrini 2007; Grandin and Charbonneau 2008). BMH1 and BMH2 were found as high-copy suppressors of the rad53-AT mutant, which is deficient in Rad53 autophosphorylation and activation (Usui and Petrini 2007). The genetic interaction led to the identification of a physical association between Rad53 and Bmh1 (or Bmh2) and to the model that Bmh1 facilitates the Rad53 activation by a direct interaction. Similarly, we not only showed that dbf4-N!109 is synthetic sick with bmh1! in the SGA screen, but also showed that the dbf4-N!109 mutant 226 eliminates the Dbf4-Bmh1 interaction in yeast two-hybrid assays. These results suggest that the Dbf4 N-terminus functions in parallel with Bmh1 and Rad53 to promote genome integrity. In this context, the dbf4-N!109 bmh1! double mutant synthetically affects the Rad53 activation and thus shows reduced fitness and additive benomyl sensitivity. Maintenance of replication-fork integrity ssDNA-RPA intermediates are also generated by stalled replication forks with uncoupled DNA polymerases and helicases (Branzei and Foiani 2009). The surveillance mechanism of the DNA replication checkpoint (also known as the Sphase checkpoint) regulates the activity of replication origins and prevents the collapse of replisomes when S-phase progression is postponed during replication stress. Rad53 directly phosphorylates Dbf4 and Sld3, which are required for activating the Mcm2-7 helicase, and consequently inhibits late origin firing (Duch et al. 2010; Lopez-Mosqueda et al. 2010; Zegerman and Diffley 2010). We recently showed that the dbf4-N!109 sld3 double mutant, which is defective in the Dbf4-Rad53 interaction and the Rad53-mediated Sld3 phosphorylation, bypasses the replication checkpoint to allow late origin firing in the presence of HU (Chen et al. 2012). Although these results imply that the Rad53 kinase binds to and phosphorylates Dbf4 to prevent late origin firing, the dbf4-N!109 mutation alone does not interfere with early origin firing or display any genotoxic sensitivity (Gabrielse et al. 2006). 227 In the SGA analysis, the dbf4-N!109 allele was found to be synthetically lethal or sick with the null mutants of the Top3-Sgs1-Rmi1 complex, which is known to control the stability of replication forks and the recovery from checkpoint arrest (Hegnauer et al. ; Cobb et al. 2005; Hegnauer et al. 2012; Yang et al. 2012). Intriguingly, the Rad53 kinase also plays a crucial role in preserving fork integrity (Labib and De Piccoli 2011). Aberrant replication intermediates and reversed forks are generally found in rad53 mutants. Though the mechanism of fork stabilization is not clear, recent studies showed that the Sgs1 subunit has separable roles in the Rad53 activation (Hegnauer et al. 2012). A defect in Sgs1related Rad53 activation might contribute to the synthetic effect in the dbf4 sgs1 double mutants. Arguing against a redundant role for Dbf4 in Rad53 activation, Mrc1 (Claspin in mammals), which functions together with Sgs1 in the Rad53 activation (Labib and De Piccoli, 2011), was not isolated in our SGA screen. However, either Rad24 or Rad9 can act in parallel to the Mrc1 function (Bjergbaek et al. 2005), and both of them were identified in dbf4 SGA studies. In addition, deletion of the POL32 gene (Figure 28B), the smallest subunit of DNA polymerase delta also targeted by the replication checkpoint, exhibits synergistic fitness defects with dbf4-N!109. We thus conclude that the Rad53 activation for replication stalling crucially relies on the complex crosstalk of Rad24, Rad9, Sgs1, and Dbf4 signaling pathways. 228 Rad53-mediated histone homeostasis Like Bmh1 and Bmh2, the histone chaperone Asf1 was found to genetically and physically interact with the Rad53 checkpoint kinase (Jiao et al. 2012). Rad53 has a crucial role in controlling histone levels and that this regulation is independent from classic Rad53 activation upon DNA damage or replication stress (Hu et al. 2001; Singh et al. 2009). Because histone deposition is coordinated with DNA replication during S phase, imbalanced histone synthesis causes cytotoxic effects, such as genomic instability and chromosome missegregation. The direct interaction between Rad53 and Asf1 is thought to link the surveillance mechanism to histone metabolism. In addition, a recent study showed that DDK phosphorylation on histone H3-Thr45 is responsive to the replication stress (Baker et al. 2010). The finding that dbf4-N!109 is synthetically sick with the asf1 null mutant suggests that Dbf4 is operating in concert with the Rad53-Asf1 complex to regulate histone dynamics during DNA replication. Interestingly, only the components of the HIR complex, and not the CAF-1 complex, are synthetic sick with dbf4-N!109. These results provide an exciting insight that Dbf4 specifically participates in a pathway that responds to Asf1-HIR involved histone regulation and Rad53 activation. MATERIALS AND METHODS Plasmids, Yeast strains, and media Yeast strains and primers used for strain construction are listed in Tables 13 and 14. PJ69–4a cells (MATa trp1-901 leu2-3,112 ura3-52 his3-200 gal4" gal80" 229 LYS2::GAL1-HIS3 GAL2-ADE2 met::GAL7-lacZ) were used for two-hybrid experiments. All other strains were derivatives of W303-1A (MATa ade2-1 trp1-1 can1-100 leu2-3, 112 his3-11, 15 ura3). Strains with the deletion of non-essential genes were made by the method of Longtine (Longtine et al. 1998). The natMX4 cassette flanked with DBF4 target sequences was PCR amplified from p4339 with primers (5’-CTA TCA ACG GCA ATG TTA TTG AAT CAC TTT CTC ATT CAC CCT TGT ACA TGG AGG CCC AGA ATA CC-3’) and (5’- ATG CAA TTG ATA ATA TAT GGA CGA GTA AAT AAG AGT TAA GTC AAT CAG TAT AGC GAC CAG CAT TC-3’) (Goldstein and McCusker 1999), and transformed into M1261 (W303 dbf4-N!109). clonNAT (Werner Bioagents) resistant transformants were confirmed with natMX4 marker and then backcrossed to W303. The dbf4!N109-natMX4 allele was PCR amplified from the genomic preparation of M3120 (W303 dbf4-#N109-natMX4) by primers Dbf4-genomic5F/R, and then integrated into M3052 (Y5565). Yeast deletion strains derived from BY4741 (MATa his3!1 leu2!0 met15!0 ura3!0) and generated by the S. cerevisiae deletion consortium were maintained in an ordered array on agar plates at a density of 1536 strains (384 unique strains arrayed in quadruplets) per plate and manipulated robotically with a colony arrayer (Bio-Rad) (Tong et al. 2001). For the synthetic genetic screening, yeast sporulation was performed using medium 2% agar, 1% potassium acetate, 0.1% yeast extract and 0.05% glucose, supplemented with uracil, histidine and leucine. Filter-sterilized solutions of Lcanavanine (50 mg/l; Sigma), G418 (200 mg/l; Invitrogen Life Technologies) and 230 clonNAT (100 mg/l; Werner Bioagents) were added to cooled media where indicated. In cases where synthetic complete medium (Scm) was supplemented with clonNAT or G418, the ammonium sulfate was replaced with monosodium glutamate and the medium termed Scm/MSG (0.17% yeast nitrogen base without amino acids and ammonium sulfate, 0.1% monosodium glutamic acid, 0.2% amino acid add back, 2% glucose and 2% agar). Benomyl (Sigma) was added directly to plates immediately before pouring (final 0.2% DMSO (v/v)). Plasmids used in this study are listed in Table 15. Deletions and point mutations within DBF4 were generated by site-directed mutagenesis using the QuikChange system (Stratagene). BMH1 (2 to 267) and BMH2 (2 to 273) were PCR amplified from genomic DNA and cloned into the EcoR1-PstI sites of pGAD-C1 to give the GalAD-Bmh1 and GalAD-Bmh2 fusions. Synthetic lethal screen and data analysis Genome-wide synthetic lethal screens were performed using synthetic genetic array (SGA) analysis as described previously (Parsons et al. 2004; Tong et al. 2004). Colonies of double-mutant progeny were photographed by using a highresolution digital imaging system developed from S&P Robotics, Inc. The colony sizes were compared to a reference set of wild-type controls. A synthetic lethal or sick interaction is determined when the colony size of double-mutant progeny is smaller than that of wild-type controls. 231 Positive hits were sorted by Gene Ontology (GO) to annotate their molecular function and biological process. The programs FunSpec and FunAssociate were used to assist functional annotations (Robinson et al. 2002; Berriz et al. 2003). Genes not falling into any category were designated as unknown function. References for all genes in this study can be found at the Saccharomyces Genome Database (SGD; (http://www.yeastgenome.org), the Yeast Proteome Database (YPD; http://www.proteome.com) and the Comprehensive Yeast Genome Database (CYGD) at MIPS (http://mips.gsf.de). All genetic interaction data is available at the General Repository for Interaction Datasets (GRID; http://biodata.mshri.on.ca/grid). Two-hybrid Analysis Various DBF4 bait constructs containing Gal4 DNA binding domain were transformed with Gal4 activation domain prey plasmids in PJ69-4a and selected on Scm plates lacking tryptophan and leucine. These were spotted at ten-fold serial dilutions on the same plates and also on plates also lacking histidine but containing 2 mM 3-aminotriazole (3AT) at 30°C and cultured for 2-3 days. 232 Table 13. Yeast strains Stain PJ69-4A W303-1A M1261 M1800 M3446 M3447 M3448 M3449 M3496 M3497 M3498 M3499 M3561 M3562 M3563 M3593 M3597 M3599 M3890 M3943 M4004 M4007 M4154 Genotype MATa trp1-901 leu2-3, -112 ura3-52 his3-200 gal4! gal80! LYS2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZ MATa ade2-1, ura3-1 his3-11, -15 trp1-1 leu2-3, -112 can1-100 W303 MATa dbf4-N!109 W303 MAT! dbf4-N!109-kanMX6 W303 MATa hir1!::HIS3 W303 MATa hir2!::URA3 W303 MATa hir3!::HIS3 W303 MATa asf1!::TRP1 W303 MATa hir1!::HIS3 dbf4-N!109::kanMX6 W303 MATa hir2!::URA3 dbf4-N!109::kanMX6 W303 MATa hir3!::HIS3 dbf4-N!109::kanMX6 W303 MATa asf1!::kanMX4 dbf4-N!109::kanMX6 W303 MATa ctf18!::HIS3 W303 MATa ctf8!::HIS3 W303 MATa dcc1!::HIS3 W303 MATa ctf18!::HIS3 dbf4-N!109::kanMX6 W303 MATa ctf8!::HIS3 dbf4-N!109::kanMX6 W303 MATa dcc1!::HIS3 dbf4-N!109::kanMX6 W303 MATa dbf4-N!109-natMX4 W303 MAT! top3-2::HIS3 sgs1-3::TRP1 W303 MATa bmh1!::kanMX4 W303 MATa bmh2!::kanMX4 W303 MATa bmh1!::kanMX4 dbf4-N!109::kanMX6 233 Source James et al., 1996 Thomas and Rothstein, 1989 Cabrielse et al., 2006 Miller et al., 2009 Sharp et al., 2005 Sharp et al., 2005 Sharp et al., 2005 Sharp et al., 2005 This study This study This study This study This study This study This study This study This study This study This study Mullen et al., 1999 This study This study This study Table 13. (cont’d) M4171 M4198 M4202 M4206 M4210 M4214 M4222 M4230 M4284 M4288 M4292 M4296 M4300 M4308 M4316 M4320 M4336 M4340 M4344 M4348 M4352 M4356 M4380 M4384 M4388 M4392 W303 MATa bmh1!::kanMX4 rad53-1 W303 MATa rmi1!::kanMX4 W303 MATa sgs1!::kanMX4 W303 MATa rad17!::kanMX4 W303 MATa rad24!::kanMX4 W303 MATa rad17!::kanMX4 dbf4-N!109::natMX4 W303 MATa rad24!::kanMX4 dbf4-N!109::natMX4 W303 MATa sgs1!::kanMX4 dbf4-N!109::natMX4 W303 MATa bch1!::kanMX4 W303 MATa cdc73!::kanMX4 W303 MATa ctk1!::kanMX4 W303 MATa lsm7!::kanMX4 W303 MATa bch1!::kanMX4 dbf4-N!109::natMX4 W303 MATa cdc73!::kanMX4 dbf4-N!109::natMX4 W303 MATa ctk1!::kanMX4 dbf4-N!109::natMX4 W303 MATa lsm7!::kanMX4 dbf4-N!109::natMX4 W303 MATa rad54!::kanMX4 W303 MATa srb2!::kanMX4 W303 MATa yta7!::kanMX4 W303 MATa rad54!::kanMX4 dbf4-N!109::natMX4 W303 MATa srb2!::kanMX4 dbf4-N!109::natMX4 W303 MATa yta7!::kanMX4 dbf4-N!109::natMX4 W303 MATa bub3!::kanMX4 W303 MATa ckb2!::kanMX4 W303 MATa tim18!::kanMX4 W303 MATa bub3!::kanMX4 dbf4-N!109::natMX4 234 This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study Table 13. (cont’d) M4396 M4400 M4404 M4408 M4412 M4416 M4420 M4422 M4424 M4428 M4432 M4436 M4440 M4444 M4494 M4498 M4501 M4505 M517 Y5565 M3130 BY4741 W303 MATa ckb2!::kanMX4 dbf4-N!109::natMX4 W303 MATa tim18!::kanMX4 dbf4-N!109::natMX4 W303 MATa rpl21b!::kanMX4 W303 MATa opi9!::kanMX4 W303 MATa sgs1-3::TRP1 W303 MATa top3-2::HIS3 W303 MATa sgs1-3::TRP1 dbf4-N!109::natMX4 W303 MATa top3-2::HIS3 sgs1-3::TRP1 dbf4-N!109::natMX4 W303 MATa rpl21b!::kanMX4 dbf4-N!109::natMX4 W303 MATa opi9!::kanMX4 dbf4-N!109::natMX4 W303 MATa csm1!::kanMX4 W303 MATa hpc2!::kanMX4 W303 MATa csm1!::kanMX4 dbf4-N!109::natMX4 W303 MATa hpc2!::kanMX4 dbf4-N!109::natMX4 W303 MATa med1!::kanMX4 W303 MATa med1!::kanMX4 dbf4-N!109::natMX4 W303 MATa pol32!::kanMX4 W303 MATa pol32!::kanMX4 dbf4-N!109::natMX4 W303 MATa rad53-1 MAT! can1"::MFA1pr-HIS3 mf#1"::MF#1pr-LEU2 lyp1" ura3"0 leu2"0 his3"1 met15!0 MAT! can1"::MFA1pr-HIS3 mf#1"::MF#1pr-LEU2 lyp1" ura3"0 leu2"0 his3"1 met15!0 dbf4-N!109-natMX4 MATa his3!1 leu2!0 met15!0 ura3!0 235 This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study Cabrielse et al., 2006 Boone Lab This study Open Biosystems Table 13. (cont’d) M3429 M3431 M3432 M3433 M3435 M3437 M3438 M3439 M3440 M3441 M3445 M3450 M3451 M3454 M3455 M3458 M3903 M3904 M4185 M4186 M4187 M4188 M4189 M4191 M4192 M4193 Open Biosystems Open Biosystems Open Biosystems Open Biosystems Open Biosystems Open Biosystems Open Biosystems Open Biosystems Open Biosystems Open Biosystems Open Biosystems Open Biosystems Open Biosystems Open Biosystems Open Biosystems Open Biosystems Open Biosystems Open Biosystems Open Biosystems Open Biosystems Open Biosystems Open Biosystems Open Biosystems Open Biosystems Open Biosystems Open Biosystems BY4741 MATa bub3!::kanMX4 BY4741 MATa hir2!::kanMX4 BY4741 MATa asf1!::kanMX4 BY4741 MATa sgs1!::kanMX4 BY4741 MATa tim18!::kanMX4 BY4741 MATa top3!::kanMX4 BY4741 MATa ctf8!::kanMX4 BY4741 MATa pol32!::kanMX4 BY4741 MATa rad17!::kanMX4 BY4741 MATa csm1!::kanMX4 BY4741 MATa dcc1!::kanMX4 BY4741 MATa lsm7!::kanMX4 BY4741 MATa rad54!::kanMX4 BY4741 MATa rmi1!::kanMX4 BY4741 MATa ctk1!::kanMX4 BY4741 MATa rad24!::kanMX4 BY4741 MATa bmh1!::kanMX4 BY4741 MATa bmh2!::kanMX4 BY4741 MATa bch1!::kanMX4 BY4741 MATa ylr235c!::kanMX4 BY4741 MATa cdc73!::kanMX4 BY4741 MATa rpl21b!::kanMX4 BY4741 MATa ckb2!::kanMX4 BY4741 MATa cbc2!::kanMX4 BY4741 MATa hpc2!::kanMX4 BY4741 MATa yta7!::kanMX4 236 Table 13. (cont’d) M4194 M4195 M4196 M4258 M4259 M4260 Open Biosystems Open Biosystems Open Biosystems Open Biosystems Open Biosystems Open Biosystems BY4741 MATa med1!::kanMX4 BY4741 MATa srb2!::kanMX4 BY4741 MATa opi9!::kanMX4 BY4741 MATa lea1!::kanMX4 BY4741 MATa pat1!::kanMX4 BY4741 MATa pfk2!::kanMX4 237 Table 14. Primers Primer Name BCH1-kanMX-F BCH1-kanMX-R BMH1-EcoRI-F BMH1-PstI-R BMH1-kanMX-F BMH1-kanMX-R BMH2-EcoRI-F BMH2-PstI-R BMH2-kanMX-F BMH2-kanMX-R BUB3-kanMX-F BUB3-kanMX-R CDC73-kanMX-F CDC73-kanMX-R CKB2-kanMX-F CKB2-kanMX-R CSM1-kanMX-F CSM1-kanMX-R CTK1-kanMX-F CTK1-kanMX-R Dbf4-genomic5F Dbf4-genomic5R DBF4-natR-F DBF4-natR-R Sequence (5’-3’) GACCCAAAGTCTATGTGAATG GATATTTGAGTAAAGCTGATC GATCGAATTCATGTCAACCAGTCGTG GCTCTGCAGTTACTTTGGTGCTTCAC CGGTGGCAAATAGCTTCCTC GAAGCTAAAGTTGCTTCTCGC GATCGAATTCATGTCCCAAACTCGTG GCTCTGCAGTTATTTGGTTGGTTCAC GTCGGTCGAAAGGGGCAAATG GAAAATTACTACTCAATTACTC GTCACCAGAAAACTCCAGTG GAGCTCTATCGCTTTATCGT GCGATGTAAAGTATAAAGTG CTTATGGAGGTATTACAAAATTG GTATATTGTTTTATGAAGAC CCAATAATTCGTGGGTAACC CAATTTTACGAATTATTTAC GGGCAACAAGAAGCAGAAGC GTGAAGCTCTATTTTTTTCG GTTGGTTGATAGGTAGTTAC CCAAATCCGTCCCACTAATAGTTTC CTTAGCCAAATCCTCCACCAAG CTATCAACGGCAATGTTATTGAATCACTTTCTCATTCACCCTTGTACATGGAGGCCCAG AATACC ATGCAATTGATAATATATGGACGAGTAAATAAGAGTTAAGTCAATCAGTATAGCGACCA GCATTC 238 Table 14. (cont’d) HPC2-kanMX-2F HPC2-kanMX-R LSM7-kanMX-F LSM7-kanMX-R MED1-kanMX-F MED1-kanMX-R OPI9-kanMX-F OPI9-kanMX-R POL32-kanMX-F POL32-kanMX-R RAD17-kanMX-F RAD17-kanMX-R RAD24-kanMX-F RAD24-kanMX-R RAD54-kanMX-F RAD54-kanMX-R RMI1-kanMX-F RMI1-kanMX-R RPL21B-kanMX-F RPL21B-kanMX-R SGS1-kanMX-F SGS1-kanMX-R SRB2-kanMX-F SRB2-kanMX-R TIM18-kanMX-F TIM18-kanMX-R YLR235C-kanMX-F YLR235C-kanMX-R CCCGCTGTTTCCCTCTCCCTC GTGGATAAAAACGAATCTC CTGTACGGACCAATTCACTC GGAATCAGTAAATAATTAAG GAAAAAATTTTTTTTCTCAAGC CCTCCTACCTACCTATCTAC GGTAGTGGTGGTGGAGGCGG CGGTTTGTCCGCTACATTGC GAAACCGAGCGGCGCTAAGC GGGATGACGCTGATGAAAAAAG CTACAAGATGGTACTGGATG CATTGATCAAGGTTGCTGATG CCTTCGTTTCATGCTCAG CGTTAGACAAAGCTTGAAG GCAAAGGGGAAGACCCTTCCG CTTGCCATAATCTTTTTTGGC GTCCTCTTGACAGGTCCGGC GTTTAGTATCTGGTCCGAGTG CTGCAGCAACGATGCTTTTTC CAGACATTGATGTTTTAAATAC GAAGCTTCTCTCCACATGTCC CTGTAGAAGAAATTGCGAACG GTGCGTTATCTACTGGGAG CTACACCAGGAACCCCGCCC CATATATGTTTCGAAGAAATC CATCATTAAAGAAACAAAAAGC GAATTGTATCTCACATATATACC CAGGTCTCGTAGTCCTAGAGAG 239 Table 14. (cont’d) YTA7-kanMX-F YTA7-kanMX-R GTTGAGGCATTAGCCGCTG GATGAATCAGCAGAGTATTC 240 Table 15. Plasmids Plasmid p4339 pCG53 pCG60 pCG63 pCG64 pCG101 pCM21 pGAD-C1 pGAD-Cdc5.3 pGBKT7 pJK117 pJK119 pYJ1 pYJ2 pYJ3 pYJ4 pYJ5 pYJ6 pYJ7 pYJ8 pYJ9 pYJ30 pYJ38 pYJ308 pYJ332 pYJ368 pYJ372 Description pCRII-TOPO::natRMX4 pGBKT7-DBF466-227 pCG53ADH1 promoter-!(-732)-(-802) pCG60 W202E pCG60 W202A pCG60 GA159,160LL pCG60-DBF466-109 pGAD-C1-CDC5421-705 pGAD-C1-BMH12-267 pGAD-C1-BMH22-273 pCG60-DBF4N!71 pCG60-DBF4N!77 pCG60-DBF4N!81 pCG60-DBF4N!87 pCG60-DBF4N!93 pCG60-DBF4N!99 pCG60-DBF4N!103 pCG60-DBF4N!107 pCG60-DBF4N!109 pCG60 R83E pCG60-DBF4!82-88 pGAD-C1-RAD531-300 pCG60-DBF4!100-109 pCG60-DBF466-190 pCG60-DBF466-150 241 Source Goldstein and McCusker, 1999 Miller et al., 2009 Miller et al., 2009 This study This study This study Miller et al., 2009 James et al. 1996 Miller et al., 2009 Clontech This study This study Chen and Weinreich, 2010 Chen and Weinreich, 2010 Chen and Weinreich, 2010 Chen and Weinreich, 2010 Chen and Weinreich, 2010 Chen and Weinreich, 2010 Chen and Weinreich, 2010 Chen and Weinreich, 2010 Chen and Weinreich, 2010 Chen and Weinreich, 2010 Miller et al., 2009 This study This study This study This study ACKNOWLEDGMENTS We thank FuJung Chang (Weinreich Lab), Ermira Shuteriqi (Boone Lab), and Nydia van Dyk (Boone Lab) for technical help or advice. 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Mol Cell Biol. 255 CHAPTER 5 CONCLUSIONS AND OUTLOOK This chapter was taken partly from the manuscript currently in preparation: Dbf4, a multifaceted cell-cycle regulator Ying-Chou Chen and Michael Weinreich 256 CONCLUSIONS AND OUTLOOK RECENT INSIGHTS INTO DDK Dbf4 is a regulator of chromosome segregation Until recently, our knowledge about the role of the Dbf4-Cdc7 kinase in cell cycle regulation has expanded from the initiation of DNA replication to the complex control of chromosome segregation in mitosis and meiosis. Our studies have highlighted the direct interaction between Dbf4 and Cdc5 and indicated that Dbf4 inhibits Cdc5 function during mitotic exit (Miller et al. 2009; Chen and Weinreich 2010). The C-terminal truncation of dbf4 mutants that are defective in Cdc7 binding bypassed the Dbf4mediated Cdc5 inhibition, suggesting that Dbf4 functions as a scaffold for Cdc7 to regulate Cdc5. Indeed, the molecular basis for the Dbf4-Cdc5 interaction was subsequently characterized as a non-canonical binding mechanism between the Dbf4 N-terminus and Cdc5 Polo-box domain (PBD), suggesting a specific role of Dbf4, together with Cdc7, in Cdc5-related cell cycle regulation. FEAR, MEN, and SPoC Among multiple roles of Cdc5 in mitosis, it has been shown that Dbf4 is functionally linked to the FEAR and MEN pathways. The segregation of highly repetitive ribosomal DNA (rDNA) on chromosome III of budding yeast depends on the activation of Cdc14 phosphatase via the FEAR pathway (D'Amours and Amon 2004). Recent studies found that stabilization of Dbf4 by removing the N-terminal D-boxes (dbf4-N!65) led to the delay of rDNA segregation and that this dbf4 mutant was synthetically lethal with the 257 cdc5-1 hypomorphic mutant (Sullivan et al. 2008; Miller et al. 2009; Chen and Weinreich 2010). More interestingly, dbf4 mutants (dbf4-N!109 or -!82-88) that abolish the Dbf4Cdc5 interaction suppressed the temperature sensitive phenotype of cdc5-1 (Miller et al. 2009; Chen and Weinreich 2010), implying that elevated levels of Dbf4 inhibit Cdc5 function in Cdc14 activation in the FEAR pathway. It should be noted, however, that neither dbf4-N!109 nor dbf4-!82-88 allows Cdc5 to induce premature Cdc14 activation (Miller et al. 2009). Because Cdc5 also activates Cdc14 though the MEN pathway, we examined the genetic interactions between dbf4-N!109 and other MEN mutants and found that dbf4-N!109 suppresses the temperature sensitivity of dbf2-1 (one of essential kinases in the MEN signal transduction), implying a closer functional connection between Dbf4 and the MEN pathway (Miller et al. 2009). Apart from cdc5-1 and dbf2-1, none of the other genes in the MEN pathway has a genetic interaction with DBF4. In addition, the dbf4-!82-88 and -R83E mutants, which specifically lose the Cdc5 interaction, cannot suppress the temperature sensitivity of dbf2-1 (unpublished data, Weinreich lab), suggesting that the Dbf4 N-terminus may also participate in the MEN pathway though a Cdc5-independent mechanism. Correct orientation of the mitotic spindle is vital for faithful chromosome segregation (Bloecher et al. 2000; Pereira et al. 2000). In S. cerevisiae, a surveillance mechanism known as the spindle position checkpoint (SPoC) modulates MEN signaling to restrain mitotic exit when the spindles are misaligned (Fraschini et al. 2008). In normal mitotic exit, Cdc5 antagonizes a two-subunit GAP (GTPase activating protein, comprising Bub2 and Bfa1), which inhibits the Tem1 kinase and subsequently prevents the kinase 258 cascades in the MEN pathway (Hu et al. 2001; Lee et al. 2001; Hu and Elledge 2002; Ro et al. 2002; Geymonat et al. 2003). Therefore, when the SPoC gets activated, it is known that Cdc5 is down-regulated to allow the Bub2/Bfa1-mediated inhibition of Tem1. We first proposed that Dbf4 inhibits Cdc5 in the MEN pathway and that the defect of the dbf4-N!109 mutant in the SPoC activation was due to loss of the Dbf4-Cdc5 interaction (Miller et al. 2009). However, the dbf4-!82-88 mutant has an intact SPoC (unpublished data, Weinreich lab). This suggests that instead of inhibiting Cdc5 by a direct interaction, Dbf4 is likely responsible through a distinct mechanism in SPoC activation. Meiotic recombination and mono-orientation Studies in yeast meiosis indicate that inactivation of the Cdc7 kinase using temperaturesensitive or analog-sensitive mutants results in pleiotropic effects on chromosome dynamics (Valentin et al. 2006; Wan et al. 2006; Matos et al. 2008; Wan et al. 2008). These mutants undergo DNA replication, but are arrested in prophase I with defects in meiotic recombination. This is probably because that DDK activity is required for the phosphorylation of Mer2, which facilitates the Spo11-mediated double-strand break (DSB) formation and interhomolog crossovers (also known as chiasmata) (Sasanuma et al. 2008; Wan et al. 2008). Because this recombination mechanism is crucial for accurate segregation of homologous chromosomes in meiosis I, it is also thought that the compromised DDK function triggers a checkpoint to cause the arrest before the first meiotic division. Furthermore, DDK is required for the transcriptional expression of NDT80 (Lo et al. 2008; Lo et al. 2012), which is a meiosis-specific transcriptional activator that regulates genes involved in exit from prophase I and the following meiotic 259 progression (Hollingsworth 2008). In the absence of Cdc7 kinase activity, prophase I arrest is bypassed by overexpressing NDT80. Taken together, these findings suggest multiple roles of DDK in setting up meiotic chromosome segregation. The Dbf4-dependent kinase also contributes to the mono-orientation of sister kinetochores (also known as syntely) in the first division of meiosis (Matos et al. 2008). Syntelic kinetochore attachment and monopolar chromosome segregation depend on the maintenance of centromeric cohesion and the assembly of the monopolin complex (Lee and Orr-Weaver 2001; Dudas et al. 2011). It is known that the casein kinase Hrr25 and DDK phosphorylation of Rec8 (Petronczki et al. 2006; Katis et al. 2010), which is the meiosis-specific cohesin subunit, are necessary for cleavage by separase Esp1 (Marston and Amon 2004), whereas the cleavage of the mitotic cohesin Scc1 relies on the phosphorylation of Cdc5 (Alexandru et al. 2001; Barr et al. 2004; Archambault and Glover 2009). It has long been known that the centromeric Rec8 is protected from cleavage by shugoshin until meiosis II, which ensures that sister kinetochores remain associated and segregated to the same pole (Watanabe and Kitajima 2005). Recent evidence unraveled that the Hrr25 and DDK phosphorylation of Rec8 is counteracted by the shugoshin-associated PP2A phosphatase (Rts1 in S. cerevisiae) in meiosis I (Ishiguro et al. 2010). Further, the Hrr25 kinase, the meiosis-specific protein Mam1, and two nucleolar proteins (Lrs4 and Csm1) form the monopolin complex. Cdc5 and DDK are both required for the translocation of Lrs4 and Csm1 from nucleoli to sister kinetochores, where they interact with Mam1 (Toth et al. 2000; Rabitsch et al. 2003; Petronczki et al. 2006; Dudas et al. 2011; Corbett and Harrison 2012). Indeed, DDK and 260 Cdc5 have been thought to complex together and execute a dual phosphorylation on Lrs4 for subcellular trafficking (Clyne et al. 2003; Lee and Amon 2003; Lo et al. 2008; Matos et al. 2008). Interestingly, genetic analyses indicate that cells harboring a dbf4N!109 allele, which abolishes the Dbf4-Cdc5 interaction, are synthetically sick with csm1! (Chen et al. 2012a), suggesting that DDK and Cdc5 also play in a collaborative way to assemble Csm1 in the monopolin complex. Sister-chromatid cohesion In budding yeast, the establishment of sister-chromatid cohesion strictly occurs in S phase when cohesin subunit Smc3 is acetylated by Eco1 (Ivanov et al. 2002; Rolef BenShahar et al. 2008; Terret et al. 2009). Recent studies suggest that DDK plays a crucial role in Eco1 regulation, which allows proper mitotic chromosome segregation (unpublished result, Morgan Lab, UCSF). Since cohesion establishment is highly linked to S-phase progression, independent genetic screens have identified that various genes within the complex of sister-chromatid cohesion are involved in DNA synthesis and DNA replication checkpoint, including MRC1, TOF1, CSM3, CTF4, and CTF18 (Mayer et al. 2004; Xu et al. 2007). Among these, Ctf18 associates with Ctf8 and Dcc1 to form an alternative RFC (replication factor C) complex, which not only is required to maintain the cohesion of sister chromatids (Hanna et al. 2001; Mayer et al. 2001; Pan et al. 2006) but also regulates late origin firing during replication stress (Crabbe et al. 2010). We recently identified that the ctf18, ctf8 and dcc1 null mutants are synthetically sick with dbf4-N!109 and display additive sensitivity to DNA-damaging agents and spindle 261 poison (Chen et al. 2012a). These results suggest that Dbf4 has a parallel role with Ctf18-RFC in an interplay between DNA replication and chromosome segregation. Dbf4 relays the checkpoint signal DNA replication checkpoint In the current model of DNA replication checkpoint, Dbf4 has been considered as a substrate downstream of the Rad53 kinase in response to replication fork arrest (Labib and De Piccoli 2011). It has been known that activated Rad53 phosphorylates multiple sites in the Dbf4 C-terminus and consequently inhibits the Cdc7 kinase from promoting late origin firing (Gabrielse et al. 2006; Yabuuchi et al. 2006; Duch et al. 2010; LopezMosqueda et al. 2010; Zegerman and Diffley 2010). Given an N-terminal deletion to Dbf4 residue 109, the Rad53-mediated Dbf4 hyperphosphorylation is significantly impaired, suggesting that these residues play a crucial role in the Rad53-Dbf4 interaction. By studying the molecular interaction between Dbf4 and Rad53, we recently found that Rad53 FHA domains directly bind to a Dbf4 T105-x-x-E-L motif in a phosphorylation-dependent manner, and we proposed that Rad53 interacts with Dbf4 dimers or multimers (Chen et al. 2012b). Moreover, loss of the Rad53-Dbf4 physical interaction prevents Rad53 phosphorylation of Dbf4, which allows late origin firing in the presence of HU. These data indicate that Dbf4 not only functions as a scaffold to conduct the Rad53-mediated Cdc7 inhibition, but also participates in relaying the signal through its self-assembly and sequential phosphorylation events. 262 Although it is thought that the Dbf4-Rad53 interaction relies on the phosphorylation of Dbf4 residue Thr105 for FHA domain binding (Chen et al. 2012b), the kinases involved are largely unknown. Recent evidence has shown that a majority of yeast kinases specifically recognize the residues flanking at the +1, -2, and -3 positions to their target serine or threonine, such as proline at the +1 position or arginine at the -2 or -3 position (Mok et al. 2010). The latter arginine-directed kinases account for 35 of 61 analyzed yeast kinases. Mutagenesis studies showed that neither Dbf4 Arg103 nor Pro106 is necessary for the Rad53 interaction, suggesting that they do not contribute to the phosphorylation of Thr105. In contrast, there are a limited number of acidophilic kinases in mammals that are able to selectively phosphorylate the FHA domain-recognizing sequence, Thr-x-x-Glu/Asp, including Polo-like kinase 1 (Cdc5 in S. cerevisiae), CK2 (Cka1), and GSK3 (Mck1, Mrk1, and Rim11) (Fiol et al. 1988; Songyang et al. 1996; Johnson et al. 2007). However, the relative importance of these kinases in replication checkpoint will require further studies. In addition to regulating the firing of late origins, Dbf4 has also been implicated in preserving replisome stability when replication forks stall. Synthetic lethal screens (SGAs, synthetic genetic assays) found that the function of Dbf4 (or Cdc7) is partially redundant with the Top3-Sgs1-Rmi1 complex, which is critical for maintaining genomic integrity by preventing the accumulation of aberrant replication or recombination intermediates (Tong et al. 2004; Chang et al. 2005; Pan et al. 2006). The DNA helicase Sgs1 (BLM in humans) has multiple roles in checkpoint signal transduction, including the binding and activation of the Rad53 checkpoint kinase (Myung et al. 2001; 263 Bjergbaek et al. 2005; Cobb et al. 2005; Hegnauer et al. 2012). Similarly, Rmi1 is required for normal fork progression and stalled fork recovery (Yang et al. 2012). The synthetic lethal or sick interaction observed between the dbf4-N!109 allele and the null mutants of the Top3-Sgs1-Rmi1 complex indicates that the N-terminal fragment of Dbf4 functions in cooperation with the Top3-Sgs1-Rmi1 complex in response to replication perturbations (Chen et al. 2012a). Indeed, dbf4-N!109 is unable to interact with Rad53 and is synthetically lethal with the rad53 kinase-defective mutant, rad53-1 (equivalent to the rad53-11 mutant) (Gabrielse et al. 2006; Chen et al. 2012b), suggesting that the Dbf4 associates with activated Rad53 at stalled replication forks to promote checkpoint responses. In support of this idea, it has been shown recently that human Dbf4 and Chk2 (Rad53 in budding yeast) are both direct targets of ATM and ATR kinases (Mec1 and Tel1) in activating the DNA replication checkpoint (Lee et al. 2012). DNA damage checkpoint One recent study in fission yeast showed that Hsk1 (Dbf4-dependent kinase) phosphorylates the PCNA-like 9-1-1 clamp (composed of Rad9, Rad1, and Hus1; known as Ddc1-Rad17-Mec3 in S. cerevisiae) in response to DNA damage (Furuya et al. 2010). The heterotrimeric complex, together with its clamp loader (Rad24 in budding yeast), plays crucial roles in recognizing DNA damage and recruiting DNA repair enzymes. The 9-1-1 complex also serves as a platform for ATR- and ATM-mediated checkpoint activation via binding to replication protein A coated single-stranded DNA (ssDNA-RPA) (Harper and Elledge 2007). DDK-mediated phosphorylation facilitates the disassociation of the 9-1-1 clamp from ssDNA-RPA intermediates and is required for 264 subsequent DNA repair. Even though a two-hybrid interaction between Dbf4 and the 91-1 clamp or clamp loader Rad24 has not been detected so far (unpublished data, Weinreich lab), it is possible that the budding yeast DDK and the 9-1-1 clamp associate by co-localizing to DNA damage loci. Consistently, it has been recently shown that DDK phosphorylation on histone H3-Thr45 is critical for the DNA damage responses in the S phase (Baker et al. 2010). In addition, deleting genes of the 9-1-1 complex produces synthetic sickness with dbf4-N!109 in the SGA screens (Chen et al. 2012a), suggesting a synergistic role between Dbf4 and 9-1-1 in the response to DNA damage. Synthetic genetic interactions were also observed between dbf4 (or cdc7) and several genes (mec1, rad53, chk1, and rad9) required for DNA damage checkpoint activation (Tong et al. 2004; Gabrielse et al. 2006; Chen et al. 2012b), suggesting the possibility that DDK indirectly modulates a signaling pathway downstream the 9-1-1 function. Together with DNA replication machinery components (Pol32, Ctf4, and the Ctf18-RFC complex), DNA repair genes (Rad52, Rad54, and Sgs1), and the yeast 14-3-3 protein (Bmh1), these defined pathways form the genetic network of DBF4 in DNA damage responses (Chen et al. 2012a). Checkpoint adaptation It was been proposed that Dbf4 functions as a molecular scaffold to coordinate three essential kinases, Rad53, Cdc7, and Cdc5, in cell cycle regulation (Miller et al. 2009; Chen and Weinreich 2010; Chen et al. 2012b). It has become clear that the association of Rad53-Cdc7-Dbf4 participates in checkpoint activation in response to genotoxicity or replication stress, whereas the Cdc5-Cdc7-Dbf4 interaction is involved in various mitotic 265 and meiotic controls. However, the biological relevance of such a ternary complex (Rad53-Cdc5-Cdc7-Dbf4) remains to be demonstrated. Recent studies in checkpoint adaptation in budding yeast and higher eukaryotes provide insights into the role of Dbf4 in the convergence of checkpoint signaling and mitotic regulation. Checkpoint adaptation refers to a mechanism by which cells are unable to repair DNA lesions ultimately escape checkpoint arrest and enter mitosis (Sandell and Zakian 1993). Yeast genetic screens have identified a number of genes that are required for checkpoint adaptation, including the casein kinase II Cka1, the phosphatases Ptc2 and Ptc3, the helicase Srs2, and the Polo-like kinase Cdc5 (Pellicioli et al. 2001; Vaze et al. 2002; Leroy et al. 2003). Recent evidence indicates that Cdc5 counteracts checkpoint activation by inhibiting the Mec1 and Rad53-mediated signaling pathway (Donnianni et al. 2010; Schleker et al. 2010; Vidanes et al. 2010). It has been shown that the cdc5 kinase-defective mutant loses the ability to adapt to irreparable DSBs, and overexpression of Cdc5 accelerates checkpoint adaptation. Similar molecular mechanisms were observed by studying CDC5 homologues in Xenopus (Plx1) and mammals (Plks) (Yoo et al. 2004; Syljuasen et al. 2006; van Vugt et al. 2010). In particular, Plk1, Plk3, and Plk4 not only bind to Chk2 but also phosphorylate Chk2 (Bahassi el et al. 2002; Tsvetkov et al. 2003; Tsvetkov et al. 2005; Petrinac et al. 2009), likely leading to G2/M checkpoint termination and cell cycle re-entry. We found that the budding yeast Dbf4 simultaneously interacts with Cdc5 and Rad53 as measured by co-immunoprecipitation assays from in insect lysates (Chen et al. 2012b). 266 Interestingly, yeast two-hybrid results have shown that loss of the residues required for the Dbf4-Cdc5 interaction promoted the interaction between Dbf4 and Rad53. This result suggests that the DNA replication or damage checkpoint signaling mediated by the Dbf4-Rad53 interaction is blocked by Cdc5 binding. In contrast, the dbf4 mutant that disrupts the Rad53-Dbf4 interaction (dbf4-!100-109) did not affect the Dbf4-Cdc5 interaction, suggesting that the Cdc5-Dbf4 interaction is relatively stable than the Rad53-Dbf4 interaction. Unexpectedly, one recent report indicated that Rad53 phosphorylates the Dbf4 residue Ser84 within the Cdc5 binding site in Dbf4 (residues 83-88) after HU treatment (Duch et al. 2010). This phosphorylation on Dbf4 residue Ser84 impairs the binding affinity to Cdc5 in vitro (Chen and Weinreich 2010), suggesting that Rad53 may prevent the recruitment of Cdc5 and early checkpoint adaptation by an inhibitory phosphorylation in the Dbf4 N-terminus. CONCLUDING REMARKS Although the essential role of Dbf4 in DNA replication has been studied intensively, not much is known about how Dbf4 links the replication machinery to checkpoint response and post-replicative cell-cycle regulation. The identification of the molecular interactions and genetic networks of DBF4 has advanced our understanding of its novel functions. These studies have given rise to a prevailing view of Dbf4 serving as a scaffold to coordinate DNA synthesis, checkpoint pathways, and chromosome segregation via the direct interactions with the Cdc7, Rad53 and Cdc5 kinases. Upon replication stresses or DNA damage, Dbf4 acts in parallel with the Top3-Sgs1-Rmi1, 9-1-1, and Ctf18-RFC complexes to converge multiple checkpoint signals for the Rad53 activation. It is known 267 that Rad53 directly interacts with and phosphorylates Dbf4, and then inhibits Cdc7 in late origin firing. Dbf4 may also function as a central regulator by tethering other checkpoint components or cell-cycle regulators in response to the S-phase perturbation. In particular, a profound understanding of the interaction between Dbf4 and Cdc5 has provided insights into the roles of Dbf4 in chromosome segregation, checkpoint adaptation, and various meiotic processes. Despite these progress, further challenges remain. For example, the molecular mechanism by which Dbf4 inhibits Cdc5 in the mitotic exit network or spindle position checkpoint is not completely understood. The role of the Cdc7 kinase in the Dbf4mediated Cdc5 inhibition is also not known. Additionally, the recent discovery that Dbf4 can simultaneously associate with Cdc7, Rad53, and Cdc5 raises the question of how the ternary complex is temporally and spatially assembled in vivo. Given that these genes are evolutionarily conserved, it is tempting to speculate that they play similar roles in checkpoint response and cell cycle regulation in higher eukaryotes. Even though genome-wide synthetic lethal screens were begun to provide a global view of Dbf4 function, much work will be required to evaluate the biological outcome of such synthetic genetic interactions. The discussion here has focused on the physiological functions of Dbf4; however, it is equally important to address the potential roles of the Dbf4-interacting partners, such as Cdc5, in DNA replication and checkpoint signaling cascades. 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