“.5 .3 .v A: : . ' ”darn-n. 5%: u ‘ .i.r.3.a.wwh. 2 es 1&5“; 1.. , . ,H’!v . ._ rivmvrl 3”...“qu \zivabsiIhfi K101.“ 1...... - ~$|ib t: I... ’3...» .1135}; it 3!! ’t: uki‘... nnl .117 tar .v.t..)t . AA...- ‘ilu. . 3.1. ‘13.; a It.» .‘|\\!\ I 1‘17 at! \1 Ivy}... .391 ...I...,( a r a 201') 2-: :jit‘a‘{ i‘a/izchlgan State University This is to certify that the dissertation entitled CDC7—DBF4 REGULATES MITOTIC EXIT BY INHIBITION OF THE BUDDING YEAST POLO KINASE CDC5 presented by CHARLES THOMAS MILLER has been accepted towards fulfillment of the requirements for the PhD. degree in Cell and Molecular Biology lit/M ALI/inl/C é ”Lb Major Professor’s Signature 116‘ /M- «w 7 /é.- - (,1 a» ,' c» 7 I Date MSU is an Afiirmative Action/Equal Opportunity Employer ‘.-w-.-—-—n-g_... v-wb' .w% 7‘»...- A- .‘ .— w... PLACE IN RETURN BOX to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 5/08 K:IProleoc&Pres/CIRCIDaIeDue,indd CDC7-DBF4 REGULATES MITOTIC EXIT BY INHIBITION OF THE BUDDING YEAST POLO KINASE CDC5 By Charles Thomas Miller A DISSERTATION Submitted to Michigan State University In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Cell and Molecular Biology 2010 ABSTRACT CDC7-DBF4 REGULATES MITOTIC EXIT BY INHIBITON OF THE BUDDING YEAST POLO KINASE CDC5 By Charles Thomas Miller The Dbf4-dependent kinase (DDK) is essential for DNA replication initiation and N-terminal Dbf4 sequences are important for a robust response to replication stress in S-phase. I identified a novel function for Dbf4 in regulating events late in mitosis. This function is mediated by an - interaction between N-terminal Dbf4 sequences and the budding yeast polo kinase Cdc5. The Dbf4-Cdc5 interaction requires a functional Cdc5 polo box domain (PBD), but is independent of protein phosphorylation. Although Dbf4 mutants proceed normally through S-phase, loss of Dbf4- dependent regulation of Cdc5 causes premature nuclear segregation and ' mitotic exit in the presence of a mispositioned spindle. Dbf4 does not regulate Cdc5 localization to spindle polebodies or kinase activity, raising the possibility that Dbf4 inhibits Cdc5 by regulating kinase targeting to specific substrates. The Cdc5 polo box domain is an in vitro substrate of DDK and alanine substitution of conserved PBD residues eliminates PBD phosphorylation. These studies raise the possibility that Dbf4 may regulate Cdc5-dependent mitotic exit by phosphorylation of the P80. ACKNOWLEDGEMENTS I would like to thank a number of people without whose assistance, encouragement and friendship this work would not have been possible. Most notably, I would like to thank my mentor Dr. Michael Weinreich for agreeing to take me into his lab, for his patience while I learned the exciting and complicated fields of yeast cell cycle biology and genetics, his encouragement and critical analysis when experimental results where not as successful as anticipated and his direction and advice throughout my entire scientific training. Successful completion of this thesis was a direct result of the thoughtful training of Dr. Weinreich, and my previous training in the laboratories of Dr. David Beer and Dr. Robert Helling at the University of Michigan. To these individuals I am also deeply appreciative. As science is truly a collaborative process, and this work is no exception, I would like to thank the numerous other members of the Weinreich Lab most notably FuJung Chang, Ying-Chou Chen, Dorine Bonte and Carrie Gabrielse for their helpful discussions, suggestions and/or direct contributions of their data and ideas to this project. Most notably, I thank Carrie for her technical advice and the initial experiments upon which this project was conceived. I wish to thank my friends and colleagues in the graduate program in Cell and Molecular Biology at Michigan State University including program iii directors Dr. Sue Conrad, Dr. Kathy Meek and former assistant director Dr. Steve Triezenberg. In addition I would like to greatly acknowledge the contributions of my committee members Dr. Kathy Meek, Dr. David Arnosti, Dr. John Kaguni and Dr. Min-Hao Kuo for their valuable time and suggestions along the way. To my loving parents, whose dedication and sacrifice laid the foundation for this and all future successes, I am forever indebted. Most of all, I wish to thank my wife Tracy for her patience and understanding during this long endeavor. Without her love and support, graduate school would not have been possible. And to my three children (Mackenzie, Charlotte and Gage) for their understanding of the many long hours required to bring this project to fruition. My hope is that when they are older, they will appreciate that the time away from them was a sacrifice worth making. iv TABLE OF CONTENTS LIST OF TABLES ......................................................................... vii LIST OF FIGURES ........................................................................ viii ABBREVIATIONS .......................................................................... x |NTRODUCTION1 CHAPTER1 CDC7P-DBF4P REGULATES MITOTIC EXIT BY INHIBITING POLO KINASE ...................................................................................... 81 Abstract82 Introduction” 82 Results... . .. .........86 The Cdc5p Polo-box Domain Interacts with the Dbf4p N-terminus .............................................................................. 86 Dbf4p Directly Interacts with Polo. 91 Dbf4 Inhibits Polo Activity... 93 Cdc7p-Dbf4p Phosphorylates the PBD ...107 Dbf4p Inhibits Cdc14p Nucleolar Release ....................... 111 Dbf4p Also Inhibits the MEN Kinase Dbf2p......................116 Dbf4p Regulates a Bfa1-Bub2p Independent Polo Activity During Mitotic Exit ...................................................... 117 Dbf4 Prevents Mitotic Exit When the Nucleus is Mispositioned................................................ ..118 Discussion... . ... . .130 Defining the Interaction Between Dbf4p and Polo .............. 131 When Does Dbf4 Inhibit Polo and Mitotic Exit?... . .132 How Does Dbf4 Inhibit Polo and Mitotic Exit? ................... 138 Materials and Methods ........................................................ 139 Construction of Yeast Strains, Plasmids and Baculoviruses139 Growth Media and Cell Cycle Experiments... .146 Two-hybrid Experiments... . 1.46 Yeast Whole-cell Extract Preparation, lmmunopreCIpItatIon and Blotting ....................................................................... 147 Co-immunoprecipitation from Sf9 Insect Cells ................. 147 Protein Expression, Purification and GST—Pull Down ......... 147 KinaseAssays... 1..48 Fluorescence Microscopy ............................................. 149 Cdc5 Protein Abundance, Kinase Activity and SP8 Localization ....................................................... 149 Nuclear Segregation Assay .......................................... 150 Dbf4 Cell Cycle Abundance ......................................... 150 Acknowledgements ............................................................. 151 References153 CHAPTER2 CDCS PHOSPHORYLATION SITE SCREEN AND ISOLATION OF PHOSPHORYLATED CDC5 ISOFORMS FOR MASS SPECTROMETRY160 Abstract ........................................................................... 161 Introduction ................................................................... .....161 Results... . 1.64 Mutation of Cdc5 PBD Residues ElImInates Cdc7- Dbf4m Phosphorylation... . ...164 Mutation of Cdc5 PBD Residues Does not Affect the Dbf4- Cdc5 Interaction” ......168 Isolation of Phosphorylated Isoforrns of Cdc5 for Mass. Spectrometry... ..170 Discussion... . .. .173 Phosphorylation of Recombinant Cdc5 PBD Protein for Mass Spectrometry... .176 Materials and Methods 1.77 Construction of Yeast Strains, Plasmids and Baculoviruses177 Two-hybrid Experiments... ..178 Protein Expression and Purification 178 Kinase assays... 179 Acknowledgements ............................................................ 1 80 References182 CHAPTER 3. SUMMARY AND CONCLUSIONS................................................186 References192 vi LIST OF TABLES Table 1. Yeast Strains Used In This Study ....................................... 140 Table 2. Plasmids Used in this Study .............................................. 144 Table 3. Plasmids Used in this Study ............................................. 179 vii LIST OF FIGURES Figure 1. The budding yeast cell cycle and checkpoints ........................ 6 Figure 2. Model of budding yeast DNA replication initiation .................. 10 Figure 3. Cdc5 is a master regulator of mitotic progression .................. 18 Figure 4. Budding yeast spindle orientation during mitosis ................... 28 Figure 5. Schematic diagram of functional Dbf4 and Cdc5 sequences....32 Figure 6. The N-terminus of Dbf4p interacts with the Polo PBD ............. 88 Figure 7. Western blot of GaI4-DNA binding domain fusions to various Dbf4 N-terminal fragments for the 2-hybrid assays shown in Figure 692 Figure 8. Dbf4p residues 67-109 interact directly with the Polo PBD ....... 94 Figure 9. Some Dbf4p persists after Cdc20 activation during an unperturbed cell cycle ....................................................... ' ........ 97 Figure 10. The Dbf4 N-terminus inhibits Polo activity ......................... 100 Figure 11. Dbf4p is a Polo inhibitor.................................................104 Figure 12. Cdc7p-Dbf4p does not alter Polo kinase abundance or actIVIty108 Figure 13. Dbf4 PIR deletion does not affect Cdc5 “mobility.” or abundance... . . . . . ...............112 Figure 14. DBF4 inhibits Cdc14p nucleolar release and the MEN pathway ................................................................................... 113 Figure 15. DBF4 regulates mitotic exit independently of BFA1-BUB2....119 Figure 16. Deletion of the Dbf4 PIR does not affect rebudding in the presence of spindle poison ............................................. 123 viii Figure 17. PIR deletion decreases cell viability in the presence of spindle p0Isons124 Figure 18. Deletion of the DBF4 PIR allows exit from mitosis in cells with misoriented nucleI126 Figure 19. Deletion of the Dbf4p PIR causes decreased growth in the presence of spindle poisons ............................................ 133 Figure 20. Deletion of the Dbf4p PIR does not enhance nucleolar segregation in cdc5-1cells135 Figure 21. Substitution of PBD residues inhibits DDK phosphorylatIon of the Cdc5 PBD... 1.66 Figure 22. PBD mutations near $63OA do not influence the Dbf4-Cdc5 interaction..................................................................167 Figure 23. Substitution of PBD residues decreases GAD-Cdc5 expres3Ion169 Figure 24. PBD mutations do not affect the Dbf4-Cdc5 interaction..................................................................171 Figure 25. Substitution of PBD residues decreases GAD-Cdc5 ExpreSSIon172 Figure 26. Resolution of phosphorylated Cdc5- PBD “by PAGE... . .. 174 Figure A1. Growth curve of N-terminal Dbf4 mutants ...................................................................... 197 Figure A2. Sensitivity of initiation mutants to DNA damaging agents198 Figure A3. Dbf4 regulates Rad53 expres3Ion200 ix 3AT APC ASE1 ASK ASK1 Asy ATM ATP ATR ATRIP BCKZ BFA1 BRCA1 BRCT BUB BUBRl ABBREVIATIONS 3-aminotriazole Amino Acid Anaphase Promoting Complex Anaphase Spindle Elongation 1 Activator of S-phase Kinase Associated with Spindles and Kinetechores Asynchronous Ataxia Telangiectasia Mutated Adenosine TriPhosphate ATM and Rad3 related ATR Interacting Protein Bypass of C Kinase 2 Byr 4 Alike 1 BReast CAncer 1 BRCA1 C-Terminal domain Budding Uninhibited by Benzimidizoles Budding Uninhibited by Benzimidizoles Related 1 Celsius CDC CDE CDH1 CDK CDTl CFI1 CHKl CHR CIP1 CLA4 CLB CLN C-terminal A DAPI DBFZ DBF4 DDCZ DDK Cell Division Cycle Cell cycle Dependent Element Cdc20 Homolog 1 Cyclin Dependent Kinase Cdc10 Dependent Transcript 1 Cdc14 Inhibitor 1 Checkpoint Kinase 1 Cell cycle genes Homology Region CDK Interacting Protein 1 cm Activity dependent 4 Cyclin B-type CycLiN Carboxy-terminal Deletion 4',6-DiAmidino-2-PhenylIndole Dumbell Former 2 Dumbell Former 4 DNA Damage Checkpoint 2 Dbf4-Dependent Kinase xi DNA DPBll DSB dsDNA DTT DYN 1 E2F EDTA EGFP EGTA ER ESP1 FAR1 FEAR FHA FIN1 FITC FOBl GO DeoxyriboNucleic Acid DNA Polymerase B 11 Double Strand Break Double Stranded DNA DiThioThreitoI Dynein 1 E2 promoter (adenovirus) Factor Ethylene Diamine Tetraacetic Acid Enhanced Green Fluorescent Protein Ethylene Glycol Tetraacetic Acid Estrogen Receptor Extra Spindle Pole Bodies 1 Factor Arrest 1 Cdc14 Early Anaphase Release Fork Head Associated (domain) Filaments in between Nuclei 1 Fluorescein IsoThioCyanate FOrk Blocking less 1 Gap 0 xii G1 G2 GEF GINS GST GTP H1 HA HCI HeLa HSK1 HSL HU INK4 IP KAR9 Gap 1 Gap 2 Guanine nucleotide Exchange Factor Go Ichi Nii San (Japanese; Sld5, Psf1, Psf2 . and Psf3) Glutathione-S-transferase Guanosine Triphosphate Histidine Histone 1 HemAgglutinin HydroChloric Acid Henrietta Lacks Homolog of Cdc7 Kinase 1 Histone Synthetic Lethal Hydronyrea INhibitor of CDK4 ImmunoPrecipitation lysine KARyogamy 9 xiii KIN4 KIP KOH LTE1 MAD MAPK MCB MCC MCM MEC1 MEN mg MgCIZ MIH1 Min MMS MOBl KINase 4 KInesin related Protein Potassium Hydroxide Leucine Low Temperature Essential 1 Mitotic Mitotic Arrest Deficient Mitogen Activated Protein Kinase M/uI Cell cycle Box Mitotic Checkpoint Complex MiniChromosome Maintenance Mitosis Entry Checkpoint 1 Mitotic Exit Network milligrams Magnesium Chloride Mitotic Inducer Homolog 1 Minutes Methyl Methane Sulfonate Mps One Binder xiv MPF MPSl MRC1 MTOCs MYOZ MYPT1 NaCl NET1 ng Ni NIH NP-4O NPT N-terminal ORC p p PAGE Mitotic Promoting Factor Multipolar Spindle 1 Mediator of Replication Checkpoint 1 MicroTubule Organizing Centers Myosin 2 Myosin Phosphatase Targeting Subunit 1 Sodium Chloride Nucleolar silencing Establishing factor and Telophase nanograms Nickel National Institutes of Health Nonidet P-40 Non Permissive Temperature Amino-terminal Origin Recognition Complex protein Proline Poly Acrylamide Gel Electrophoresis XV PBD PCNA PDSl P13K PIR PLK1 POL PP1 PPZA PP4 PPH21 PPH 22 pre-RC RAD RB rDNA RPA RTS 1 Polo Box Domain Proliferating Cell Nuclear Antigen Precocious Disassociation of Sister Chromatids Phosphatidyl-inositol 3 Kinase Polo box Interacting Region Polo-Like Kinase 1 Polymerase Protein Phosphatase 1 Protein Phosphatase 2A Protein Phosphatase 4 Protein PHosphatase 21 Protein PHosphatase 22 pre-Replicative Complex RADiation sensitive RetinoBlastoma Ribosomal DNA Replication Protein A Suppressor of Rox3 Temperature Sensitive allele 1 xvi SAC SCC SCC3 SF9 SIC1 SIN SLDZ SLD3 SLI15 SLK19 SMCl SMC3 SPB SP012 SPOC ssDNA SWEl SWIS Synthesis Spindle Assembly Checkpoint Sister Chromatid Cohesion 1 Sister Chromatid Cohesion 3 Spodoptera frugiperda 9 Subunit Inhibitor of CDK Septation Initiation Network Synthetic Lethal with pr11-2 Synthetic Lethal with pr11-3 Synthetic Lethal with Ipl15 Synthetic Lethal with Kar3 19 Structural Maintenance of Chromosomes 1 Structural Maintenance of Chromosomes 3 Spindle Pole Body SPOruIation 12 Spindle Position Checkpoint Single Stranded DNA Saccharomyces WEe1 SWItching Deficient 5 xvii TEL1 TEM1 Thr TPD3 TPL tRNA ts U9 YPD Threonine Time TELomere maintenance 1 TErmination of M phase 1 Threonine tRNA Processing Deficient 3 Increase in Ploidy 1 transfer RNA Temperature Sensitive micrograms Tryptophan Wild type Yeast extract Peptone Dextrose xviii INTRODUCTION CELL CYCLE OVERVIEW Fundamentals of the Eukaryotic Cell Cycle Duplication and partitioning of the genome into two daughter cells is a fundamental process of life. Cells without a nucleus (prokaryotes) replicate by binary fission, a process where cells physically separate into two daughter cells, each with a genetic complement. Organisms containing nucleated DNA (eukaryotes) by contrast separate replication and chromosome division into the synthesis (S)-phase and mitotic (M)-phase respectively. These phases are divided by two gap (G) phases: one preceding DNA synthesis (G1) and another (G2), which separates replication from mitosis (Figure 1). The length of gap phases varies considerably between species. In general, G phases enhance cell cycle regulation and maintain genome fidelity. In multicellular organisms many differentiated cells exit the cell cycle, stop dividing or exist in a reversible state of senescence or self-induced quiescence (GO-phase). In addition, cells that sustain irreversible genetic damage can permanently exit the cell cycle as an alternative to programmed cell death (apoptosis). In the first phase of interphase (G1), cells resume normal biosynthesis after completion of the preceding cell cycle and disassemble the mitotic machinery (Figure 1). During this phase, cells grow in size and respond to external stimuli using receptors on the cell surface (i.e. receptor tyrosine kinases and G-protein coupled receptors) or within the cytoplasm (i.e. nuclear hormone receptors). In unicellular organisms, the decision to remain in G1 or to enter the cell cycle is primarily determined by the nutritional status of the environment [1, 2]. Metazoan cells, by contrast, must integrate myriad signals that influence cell fate [3]. Synthesis of enzymes required for S—phase also occurs in G1 in preparation for the highly anabolic phase of DNA replication. Transition through the restriction point/START (G1-phase) is a highly regulated and irreversible process requiring faithful replication and segregation of chromosomes. Eukaryotic S-phase commences _at the initiation of DNA replication and is complete once the entire genome has been duplicated. In general, genomic replication occurs outside actively transcribed regions [4]. Successful completion of DNA synthesis produces a homologous pair of intact sister Chromatids, which are connected at the centromere and along the chromosome arms until mitosis. Critical to the success of DNA replication is a collection of S-phase checkpoint proteins (discussed in more detail below), which respond to problems during DNA replication. After replication, cells enter G2 until the beginning of mitosis. During G2, cells synthesize proteins required for assembly of the mitotic spindle in preparation for chromosome segregation and monitor the genome for the presence of DNA damage. Damage is corrected (as discussed below) prior to the beginning of mitosis and sister chromatid separation. Mitosis is the most physically dynamic phase of the cell cycle. In mammalian cells, mitosis commences in prophase with the rising activity of mitotic kinases, inducing nuclear envelope disassembly, chromosome condensation and separation of sister chromatid arms. As chromosomes condense they attach to bipolar centrosomes and align along the metaphase plate. After cells ensure that all sister chromatids are properly attached to the mitotic spindle, rapid degradation of cohesion proteins during anaphase permits the segregation of chromatids to opposite spindle poles. In telophase, chromatids decondense and the nuclear envelope reforms. Cells physically separate by the formation of an actin cleavage furrow during cytokinesis. Constriction of the actin contractile ring minimizes the cytoplasmic volume between daughter cells and forms a cytoplasmic bridge in preparation for abscission. Molecular Regulation of the Eukaryotic Cell Cycle Faithful transmission of the genetic material is an absolute requirement of life. As a consequence, all organisms have developed a sophisticated and tightly regulated system of signaling pathways and mechanisms to ensure accurate transmission of genetic material to progeny. To understand the biochemical nature of cell cycle progression in both normal and malignant cells, considerable effort has been invested in the last four decades to identify and characterize proteins involved in regulating cell division. Seminal discoveries by Tim Hunt and Paul Nurse in sea urchins and fission yeast, respectively, identified crucial proteins regulating entry into mitosis [5, 6]. Hunt and colleagues observed cyclical expression of proteins (cyclins) during every round of mitosis in sea urchin embryos. In the laboratory of Paul Nurse, fission yeast wee1 mutants were shown to enter M-phase prematurely [6]. These and subsequent studies led to the conclusion that the activity of cyclin-dependent kinases is required for entry into mitosis and progression through the cell cycle [7-10]. A family of cyclins and cyclin-dependent kinases (CDKs) control the progression through G1 and initiation of DNA replication in mammalian cells [11]. Two broad classes of CDK inhibitors, which are regulated by growth factors, target G1 CDKs and suppress their activity [12]. The lNK4A family (p15, p16 and p19) appears to exclusively regulate Cdk4-cyclin D activity [12]. Cip1 and Kip1 (p21 and p27) are more promiscuous and inhibit the activity of multiple CDKs [13, 14]. In humans, as cells exit quiescence (GO) and enter G1 phase, expression and binding of D-type cyclins (D1, D2, and D3) to Cdk4 and Cdk6 initiates a phosphorylation cascade targeting the retinoblastoma (Rb) family of transcriptional repressors [15, 16]. Hyperphosphorylation of the Rb family of ‘pocket proteins’ facilitates their release from DNA-bound E2F transcriptional activators, which initiate the coordinated expression of cell cycle specific genes [17]. In addition, CDK phosphorylates and inactivates the Cdc20 homolog 1 (th1) subunit of the anaphase promoting complex (APC), inhibiting its ability to ubiquitinate cell cycle proteins and target them for proteosomal degradation [18, 19]. Late in G1, Cdk2-cyclin E complexes phosphorylate Rb on additional sites, reinforcing progressionthrough the restriction point and into S-phase [20]. At this moment, cells are irreversibly committed to replicating their DNA and segregating their chromosomes into two daughter cells. The unicellular fungus Saccharomyces cerevisiae has a single cyclin- dependent kinase (cell division cycle 28) that regulates entry into S-phase and progression through mitosis [21]. Activation of CDK in -budding yeast is regulated by the G1/S-phase cyclins (Clns) 1-3 and B-type cyclins (Clbs) 1-6 [22-25]. Cyclin expression and CDK activity is tightly regulated in all eukaryotic organisms by pathways that integrate nutrient and mitogenic signals from within the cell and from receptors at the cell surface [25]. In budding yeast, the primary determinant for progression through the restriction point is cell size [26]. Completion of the preceding cell cycle results in mother and daughter cells of unequal size (Figure 1). As a result, the larger mother cells progress more rapidly through the next G1 phase compared to smaller daughter cells, which take longer to reach the critical size threshold. Integrating cell size with molecular preparation for S-phase entry, newborn mother and daughter cells must reach a critical size to express a sufficient quantity of Cln3 and bypass of C kinase 2 (Bck2) to activate Swi4/6 binding factor (SBF) and MIu1-box binding factor (MBF), G1 transcription factors that stimulate transcription of Cln1, 2 and B-type cyclins [22, 25, 27-29]. Cdc28- C|n2 activity, which initiates bud emergence, precedes full activation of Cdc28- Clb5 as a result of persistent expression of the Cdc28-Clb inhibitors Sic1 [30-32]. Phosphorylation of CDK inhibitors by Cd028-Cln triggers SCF (Skp, Cullin, F- box) binding and ubiquitylation, and results in full activation of the S-phase Cdc28-Clb kinases [33, 34]. Stabilization of Cd028-Clb activity then reduces Cln expression in preparation for events later in the cell cycle [35]. Progression through START (G1 to S transition) is inhibited in the presence of mating factor, which activates a MAP kinase signaling-cascade and triggers activation of the CDK inhibitor factor arrest 1 (Far1) [36, 37]. Figure 1. The budding yeast cell cycle and checkpoints. Budding yeast enter the cell cycle in G1, following cytokinesis and the physical separation of mother and daughter cells. At this time, cells grow until they reach a critical size threshold after which they traverse START and begin DNA replication in S—phase. Coincident with DNA replication initiation is bud emergence and spindle pole body duplication. During 62 phase, cells monitor the genome for the presence of DNA damage and initiate nuclear migration towards the mother-daughter bud neck. Entry into mitosis is activated by increased catalytic activity of CDK (Cdc28-Clb) and is inhibited by morphological defects in the budding yeast. Formation of the intranuclear mitotic spindle drives nuclear division and the separation of sister chromatids into mother and daughter cells. Defects in bipolar spindle attachment and spindle orientation delay progression through mitosis until these checkpoints are satisfied. The cell cycle is complete following downregulation of CDK activity during mitotic exit and the separation of cells during abscission. \I/ 5.0.8.33 mam Q / eocemLeEw 03m owns...— m 52.0.0 0:: 350.5 :00 c 5.52.83. <20 magicao a 5.05.810 Hflqumwhm 20.20.48”. Eczemocmem oEomoEoEU 92.0.0.8...0 5:28“. \ x \ ; ...Mwmn. >..m:mmw mcficsm Events preceding DNA replication initiation, including origin. binding and helicase loading, are best understood in S. cerevisiae. This is likely attributable to the well-defined replication origin binding sequence in this organism. The hexameric origin recognition complex (Orc1-6) interacts with the A and B1 elements (~30bp DNA) of origins in budding yeast [38, 39]. The sequences required for ORC binding in other organisms are not as well-defined and are thought to depend on mechanisms other than DNA sequence [40-42]. In budding yeast, fission yeast and Drosophila, ORC binding appears constitutive throughout the cell cycle; this is in contrast to human and Xenopus cells, which indicate at least a partial clearance of ORC from origins during metaphase [43-46]. ORC binding stimulates Cdc6 and Cdt1-dependent loading of minichromosome maintenance DNA helicase proteins (Mcm2-7), forming the pre-replicative complex (pre-RC) (Figure 2) [47]. In addition to DNA sequence and chromatin elements that regulate pre-RC formation, trans-acting proteins participate in coordinating cell cycle progression with assembly of the pre-RC to prevent unauthorized replication. Cdc28-Cln and Clb kinases negatively regulate pre-RC formation by phosphorylating Cdc6, which triggers its nuclear export (mammalian cells) or degradation (yeast) [48-54]. The pre-RC inhibitor Geminin also inhibits MCM loading in metazoans independent of CDK activity [55, 56]. Two protein kinases Cdc28-Clb5 (S—phase CDK) and Cdc7-Dbf4 (Dbf4- dependent kinase; DDK) are required to trigger DNA replication initiation at origins in the budding yeast (Figure 2) [21, 57]. Phosphorylation events by CDK and DDK stimulate the assembly of additional proteins at the pre-RCs and precipitate activation of the replicative helicase Mcm2-7 and origin unwinding [58- 60]. Recent evidence suggests that the only essential proteins requiring Cdc28 phosphorylation at budding yeast origins are two initiation proteins: 8le (synthetic lethal with Dbp11-2) and Sld3 [61, 62]. Cd028 phosphorylation of $le and Sld3 facilitate their interaction with pr11, which is necessary to recruit Cdc45, GINS and replicative DNA polymerases to sites of origin unwinding (Figure 2) [61, 62]. S-phase is marked by the initiation of chromosome replication and duplication of the spindle-pole bodies (SPBs)/centrosomes. Eukaryotic DNA replication is bidirectional and semi-conservative with each strand serving as a template for a newly synthesized nascent strand [63-66]. The pace of S-phase progression is controlled by the regulation of origin firing throughout S-phase [67]. Early origins fire shortly after passage through the restriction point with late origins initiating later in S-phase [68-72]. DNA damage or low nucleotide levels initiate a delay in late origin firing to slow progression through S-phase and delay chromatid separation in mitosis [73, 74]. During every S-phase, replication forks encounter myriad genetic lesions that must be overcome for faithful duplication of the genome [75, 76]. Eukaryotes utilize a variety of polymerases for both leading and lagging-strand synthesis as well as translesion and mismatch—repair polymerases that repair and replace damaged or mismatched bases [77]. Polymerase a initiates replication at origins following helicase unwinding and prior to loading the highly processive polymerases (Pol e and 8), which coordinate leading and lagging strand synthesis [77, 78]. A primary factor Figure 2. Model of budding yeast DNA replication initiation. Initiation of DNA replication commences in late G2/M phase of the preceding cell cycle. During this period, the budding yeast initiator, ORC (origin recognition complex), binds origins of replication throughout the genome. Binding of ORC to origins allows the association of Cdt1 and Cdc6 with the initiator and facilitates MCM (minichromosome maintenance) loading on origins. The triggering mechanism for replication initiation at origins requires the catalytic activity of CDK (cyclin dependent kinase) and DDK (Dbf4-dependent kinase). Phosphorylation of $le and Sld3 by CDK is thought to trigger association with the BRCT containing protein pr11 and facilitate polymerase association with origins of replication. Genetic and biochemical evidence suggests that MCM phosphorylation by DDK triggers DNA unwinding by helicases and DNA replication initiation. 10 contributing to replication processivity is the homotrimeric clamp proliferating cell nuclear antigen (PCNA), which localizes polymerases to DNA at the replication fork [79, 80]. Studies have placed replication proteins at nuclear foci within replicating cells, suggesting that replication forks are organized at “replication factories” throughout eukaryotic chromatin [81]. This is in contrast to previous models where replication forks moved along stationary DNA. Following DNA replication, cells prepare for chromosome division by initiating mitosis. Timely entry into mitosis in eukaryotic organisms requires the activation of a critical mitotic CDK (Cd028 in budding yeast; Cdc2 in fission yeast and humans) [82]. In most eukaryotic organisms, activation of mitotic CDK requires the cooperative action of two events. First, expression of mitotic cyclins must reach a critical threshold to activate the catalytic CDK subunit [83, 84]. Secondly, reversal of Wee1 inhibitory phosphorylation on a critical CDK tyrosine residue by the Cdc25 class of phosphatases elevates mitotic CDK activity and triggers entry into mitosis [85]. The antagonistic function of the Cdc25 phosphatase and Wee1 kinase in controlling the activity of mitotic CDK is highly conserved throughout evolution. First evidence for the CDK1/mitotic promoting factor (MPF) in initiating mitotic progression was provided by the cell-fusion experiment of Rao and Johnson, which demonstrated that a diffusible factor could initiate mitosis in cells arrested in non-mitotic stages of the cell cycle [9]. Key studies in the fission yeast Schizosaccharomyces pombe identified and demonstrated the epistatic relationship between genes that delayed (cch and 12 cd025) or sped up (wee1) the cell cycle, resulting in elongated or shortened cells respectively [86, 87]. In eukaryotic organisms, high CDK levels trigger the duplication of the spindle pole body/centrosome in cells [88]. Metazoan centrosomes have dramatically different structures than yeast spindle pole bodies. Animal cell centrosomes usually comprise a pair of centrioles that serve as microtubule organizing centers (MTOCs) for the capture and segregation of chromosomes [89]. Yeast MTOCs are fundamentally different in structure and are organized as molecular plaques that remain attached to the nuclear envelope throughout a closed mitosis [90]. Animal MTOCs anchor themselves to opposite cellular poles with cortically attached microtubules and promote microtubule invasion of the cellular midzone early in mitosis in an attempt to capture and bind to sister- chromatid pairs [89]. Chromosome centromere and arm attachment is maintained throughout mitosis by a class of chromatid binding proteins called cohesins (yeast cohesin complexes contain sister chromatid cohesion 1 (Scc1), Scc3, structural maintenance of chromosomes 1 (Smc1) and Smc3) [91]. Degradation of cohesin subunit Scc1 by the protease Separase (extra spindle pole bodies 1 (Esp1)) is required for cohesin removal at the metaphase-to- anaphase transition. Inhibition of Esp1-dependent separation during mitosis is regulated by the APC-CchO substrate securin (precocious dissociation of sisters 1 (Pds1) in budding yeast) [92—94]. Activation of the APC by chromatid binding to the mitotic spindle triggers Pds1 degradation and activation of Separase [92]. In contrast to budding yeast, vertebrates remove chromosome cohesins in two 13 successive steps. Early in pro-metaphase, cohesins are removed from chromosome arms by a protease-independent mechanism requiring phosphorylation by Polo-like kinase 1 (Plk1) [95]. Cohesins located at the kinetechore are maintained until APC activation in early anaphase [92]. Sister chromatid separation in both metazoans and yeast occurs only after all kinetechores have achieved bipolar attachment to the mitotic spindle [96]. Failure of a single chromatid pair to properly align along the metaphase plate triggers stabilization of the spindle assembly checkpoint (SAC) and metaphase arrest. Sister chromatids migrate to opposite cellular poles following Cdc20 stabilization and activation of the APC [97, 98]. In contrast to metazoan chromosomes, budding yeast chromatids must traverse the mother-daughter bud neck prior to exit from mitosis to prevent stabilization of the spindle position checkpoint (described in more detail below) [96]. In budding yeast, activation of the nucleolar phosphatase Cdc14 is an important event in bringing about the resolution of mitosis following sister chromatid segregation. The first demonstration of the critical function of Cdc14 was in temperature sensitive (ts) mutants that arrested in late telophase with high CDK levels [99]. Cdc14 is a dual-specificity phosphatase and triggers mitotic exit and reentry into G1 phase [100-102]. The first well-characterized roles of Cdc14 were therefore its dephosphorylation and activation of the Cdk1 inhibitor Sic1 and its transcriptional activator, Swi5, and stabilization of the APC subunit th1 [100]. Activation of this essential function of Cdc14 in mitotic exit requires the coordinated regulation of a group of signaling proteins within the mitotic exit 14 network (MEN) (Figure 3) [102, 103]. Critical to MEN regulation is the spatial and temporal regulation of the daughter bound spindle pole body relative to the bud neck [104]. Activation of the MEN is achieved by triggering a shift in the SPB GTPase Tem1 towards an active GTP-bound state [105, 106]. Prior to MEN activation, the two-component GTPase activating protein (GAP) Bfa1-Bub2 antagonizes MEN signaling by enhancing Tem1-GTPase activity at SPBs [106]. Coordination of MEN activation with spindle elongation is regulated by bud neck localization of Lte1 during mitosis [104]. During anaphase the daughter-bound SPB passes toward a region of high Lte1 concentration (budneck), effectively switching Tem1 towards the GTP bound state. Tem1-GTP then activates the SPB kinases Cdc15 and Dbf2-Mob1 upstream of Cdc14 localization [107, 108]. Phosphorylation of Cdc14 and its inhibitor Cfi/Net1 within the nucleolus releases Cdc14 and initiates mitotic exit [109-111]. In addition to the role SPB localization plays in determining mitotic exit onset, CDK activity and activation of the Polo-like kinase Cdc5 is absolutely required for MEN activation (Figure 3) [112]. Cdc14 dephosphorylation of CDK sites on Cdc15 is required for full activation of the mitotic exit network [113]. In contrast, CDK phosphorylation is required for Cdc14 nucleolar release by directly phosphorylating nucleolar Net1/Cfi1 [110]. Cdc5 localizes to both SPBs during mitosis and phosphorylates the Bfa1 subunit of the Bfa1-Bub2 GAP [114, 115]. This triggers Bfa1-Bub2 subunit dissociation and enhances MEN signaling [116]. Deletion of BUBZ in budding yeast allows rebudding similar to mitotic arrest deficient (MAD) mutants in the presence of spindle poisons [117]. Consistent with 15 the model for Cdc5 MEN regulation, overexpression of wild type Cdc5 causes premature Cdc14 release in cells arrested in S-phase (hydroxyurea) and rebudding in cells treated with spindle poisons (nocodazole) [112, 118]. In addition, some experiments suggest an additional function for Cdc5 in regulating full Cdc14 release outside of the MEN, although these findings await further investigation [1 19]. The discovery of a parallel pathway that regulates Cdc14 localization led to the characterization of a group of factors involved in regulating events in early anaphase [120]. Transient release of Cdc14 by the Cdc14 early anaphase release (FEAR) network results in nuclear release, in contrast to full activation and cytoplasmic localization of Cdc14 by the MEN (Figure 3) [106, 121]. Cdc14 release in early anaphase is important for stabilization of elongated spindle microtubules, assembly of a spindle midzone and resolution of late segregating regions of the genome including the rDNA locus [122-125]. CDK substrates that are dephosphorylated at this time include Ask1, Ase1, Fin1 and Sli15 [126]. Chromosome XII in budding yeast contains ~150 tandem copies of the DNA encoding ribosomal subunits. Activation of the FEAR network is important for resolution of this locus during anaphase [124, 127, 128]. Key to uncovering this pathway was the observation that Cdc14 transient release is triggered by sister- chromatid resolution and phosphorylation of six sites on Cfi1/Net1 by CDK [110, 111]. CDK activity is antagonized throughout mitosis by protein phosphatase 2A (PP2A) (Figure 3) [129, 130]. PP2A is regulated by the targeting subunit Cdc55 and inhibits Cdc14 release through dephosphorylation of Cd028 Net1 phospho- I6 sites [129, 131]. Deletion of CDC55 results in cellular rebudding in the presence of spindle poisons, although to a lesser extent than deletion of BUBZ [117, 130]. Interestingly the FEAR components Esp1 and Slk19 are important for Cdc55 inactivation and Spo12 CDK phosphorylation, although the mechanism by which Spo12 and its binding partner Fob1 contribute to Cdc14 activation is not well understood [126]. The role for Cdc5 in regulating Cdc14 activity in the FEAR is even less clear (Figure 3). Epistasis analysis places Cdc5 downstream or in parallel of Esp1 and Slk19 [112, 120, 132]. Data suggesting that Cdc5 interacts with and phosphorylates Cdc14 suggests that Cch coordinates Cdc14 release with Esp1, Slk19 and CDK [133]. A recent report suggests that Cdc5 activation of the FEAR occurs indirectly through inactivation of the CDK inhibitor Swe1, thereby increasing Cdk activity on Net1 [134]. Although this model is attractive, it awaits additional experimentation. Phosphatase Regulation of the Cell Cycle The critical role protein kinases play in controlling entry and exit from the cell cycle is well established, although the essential contribution that protein phosphatases play in this process has only recently been fully appreciated. It is now understood that phosphatases are critical players in regulating progression through many of the cell cycle phases. Of all the protein phosphatases identified, only a handful have been linked to mitotic regulation by biochemical or 17 Figure 3. Cch is a master regulator of mitotic progression. The budding yeast Polo kinase Cdc5 positively regulates multiple stages of mitosis. Entry into mitosis is regulated by activation of the yeast CDK protein Cdc28. Inhibitory phosphorylation of Cd028 by Swe1 is reversed by the phosphatase Mih1 (human Cdc25 homolog) during progression into mitosis. Cdc5 antagonizes Swe1 function during mitotic initiation through its association with proteins at the mother-daughter bud neck. Progression through the metaphase-anaphase transition requires bipolar spindle attachment at sister-chromatid kinetechores which triggers stabilization of the CchO-APC, ubiquitylation of Securin (Pds1) and proteolytic cleavage of chromatid cohesins by Separase (Esp1). Cdc5 phosphorylation of the cohesin subunit Scc1 is thought to enhance Separase activity on cohesins, but is not required for entry into anaphase in the budding yeast. The essential role for Cdc5 is in the activation of mitotic exit through release of Cdc14 from its inhibitory state within the nucleolus. This occurs by positive regulation of two pathways (MEN, mitotic exit network; FEAR, cdc14 early anaphase release) that affect Cdc14 activity. Cdc14 is inhibited during much of the cell cycle by association with the inhibitor Net1 within the nucleolus. Cdc5 activity is required at the beginning of anaphase for the transient release of Cdc14 into the nucleus. The precise requirement for Cdc5 is presently unknown, but recent evidence raises the possibility it may regulate FEAR by enhancing CDK phosphorylation of Net1 or direct phosphorylation of Cdc14. In addition to Cdc5, the Esp1-Slk19 complex and protein phosphatase PP2A-Cdc55 are also required for proper Cdc14 function in the FEAR network. MEN activation of Cdc14 requires Cdc5-dependent inhibition of the MEN antagonist Bfa1-Bub2, presumably at yeast spindle pole bodies. Disruption of the Bfa1-Bub2 interaction enhances Tem1-GTP activation of Cdc15, initiating a kinase cascade that results in hyperphosphorylation of Net1 and full and sustained release of Cdc14 resulting in mitotic exit. 18 00000505. 00000000 «0 5 0000020.. 0000005. 0000000< III 32.0 :02 £80 - Sam ._>_._.wx0v_._0mz_¢4xmmuzmma>0n=m0>¥4>m>4mEmewm9E§§m.52!IIquzuOuhOowamuEgEmSu—R. ..N ..D ||||IAI||‘I|.AII‘IIIAIII‘II| us be «a E. a; an a a mum «am New >mw42§5>¥>muzmw4mmaxmw._._>Im<>>>0m¢oo>m_>>>ummtozzufi>0_owww40>wuozxzm>o>x>¥h22mz no em as a ,3 .3. mm. mm. a U m2... Equu xom o_on_ Equu own—5. I F mono m ommEmu <20 2 o>Ewccw < I come: EN cacao 5% «E II 96% no 9:23 so x36! 5 .8 .899 I I 32 m._zl 37 U E z S So one mom own at m? Enn— < 33 BRCT domains are primarily found in DNA damage-response proteins [232, 233]. Crystallization of the Brca1 tandem BRCT repeat identified the domain as a phosphopeptide-binding module [234]. Recent work in our lab identified a c- terminal region of the BRCT sequence that is conserved in Dbf4 homologs prompting us to name it the BRDF @CT and Dbf4 similar) motif [229]. However, crystallization of this domain by Guarne’ and colleagues suggests that it nonetheless adopts a canonical BRCT-fold [229, 235]. The function of the Dbf4 BRCT domain is unknown and proteins that interact exclusively with the BRCT domain have not been identified. Dbf4 is a target of Rad53-dependent checkpoint phosphorylation in response to replication stress and this regulation requires an intact Dbf4 N-terminus [229, 236]. In addition, Cds1/Rad53 kinase activation is severely perturbed in hsk1-89 cells and the hsk1-89 mutation is synthetically lethal with the checkpoint protein Rad3/Mec1 (ATR homolog in yeast) suggesting functional overlap between Cdc7-Dbf4 and the DNA damage response pathway [237]. A possible function of the Dbf4 BRCT domain is to interact with phosphorylated Rad53 FHA domain. Molecular modeling of the crystallized BRCT domain raises the possibility, but biochemical evidence for this is lacking [235]. Masai and others proposed that human Cdc7-Dbf4 interacts with components of stalled replication forks and may be required for replication restart in response to replication stress [238]. Expression and Regulation of DDK subunits during the Cell Cycle 34 Cdc7 catalytic activity is regulated by cyclical expression of Dbf4 throughout the cell cycle [239]. In contrast to its regulatory subunit, Cdc7 protein levels remain relatively constant during the yeast cell cycle [239]. Mammalian Cdc7 activity is positively regulated by serum stimulation and E2F transcription factor activity [226]. Dbf4 protein levels remain low throughout G1 and increase at the transition into S-phase and decrease at the end of mitosis [239]. Dbf4 promoter elements suggest that its transcription is under control of the Mlul cell- cycle box (MCB) transcriptional element, however the observed protein oscillation within cells appears to be regulated at the level of protein stability [240]. Dbf4 contains two putative “RXXL” destruction boxes within the protein N- terrninus (Figure 5) suggesting a role for APC-Cdc20 in regulating Dbf4 destruction. Mutation of the N-terminal destruction box can stabilize Dbf4 [241]. In addition, inactivation or mutation of genes encoding APC subunits stabilizes Dbf4 protein, however the precise regulation of Dbf4 destruction remains enigmatic [236, 242]. APC activity is regulated by the specificity components, Cdc20 and th1, which become active at the metaphase-to-anaphase transition and during mitotic exit respectively [243]. The contribution each subunit plays in targeting Dbf4 for destruction is currently unclear, however, persistent expression of Dbf4 in mitotic exit mutants (cdc5—1, dbf2-1 and cdc14-1) at non-permissive temperature argues against exclusive regulation by Cdc20 and suggests that Dbf4 levels persist late into mitosis, long after completion of S—phase [236, 242]. So how is Dbf4 stability regulated? Cdk phosphorylates Dbf4 during the normal cell cycle [244]. A serine within the N-terminal Dbf4 D-box (“RSPL”) matches a 35 consensus CDK phosphorylation site and is phosphorylated in vivo after treatment with MMS (Jaime Lopez; personal communication). It is possible that CDK actively inhibits Dbf4 destruction prior to mitotic exit when the Cdc20 APC is active. Cdc14 dephosphorylation of CDK sites raises the possibility that Cdc14 may antagonize CDK phosphorylation of Dbf4 and allow the APC access to the Dbf4 destruction box late in mitosis. Cdc7-Dbf4 Function In S-phase Initiation In contrast to our understanding of the role for CDK in replication initiation, the precise biochemical role for DDK at origins is less clear. What is apparent is that localization of DDK to origins is critical for the timely and accurate firing of replication origins throughout S-phase. Diffley and colleagues demonstrated by a one-hybrid assay that Dbf4 interacts with origins, and that this genetic interaction is dependent on the origin recognition complex ORC [39]. Additionally, studies in Xenopus indicate that DDK associates with the pre-RC at origins and this association is dependent on Dbf4 [245]. The pre-RC proteins Orc2, Orc3, Mcm2 and Mcm4 all interact with Dbf4 suggesting a model whereby Dbf4 targets Cdc7 to sites of pre-RC binding [246]. Cdc7-Dbf4 can phosphorylate multiple proteins at the origin including several Mcm subunits, Cdc45 and polymerase alpha [58, 236, 247]. Mapping of Cdc7-Dbf4 phosphorylation sites on Mcm proteins and the identification of the bob1-1 (mcm5, P83L) bypass suppressor of cdc7A and dbf4A cells led to the hypothesis that Cdc7-Dbf4 activates the Mom replicative helicase [223, 248, 249]. It is thought that phosphorylation of Mom 36 proteins by Cdc7 likely leads to a conformational shift in the helicase complex, initiating origin unwinding and DNA replication [250]. Recent evidence suggests a model whereby Cdc7 phosphorylation of MCM N-termini may trigger MCM double hexamer dissociation and DNA unwinding [251]. The mechanism whereby cells regulate the timing of origin firing throughout S—phase and delay the activity of Cdc7-Dbf4 on late origins in response to cellular stress remains to be established. Recent evidence suggests that an initiating phosphorylation event by a DDK-independent kinase primes the pre-RC Mcm2-7 complex for phosphorylation by Cdc7-Dbf4, suggesting a model in which the spatial and temporal regulation of Cdc7-Dbf4 activity on origins might be controlled [59]. Cdc5/Polo: Structure and Function Molecular understanding of the important role of Polo kinases in regulating cell cycle progression and cellular homeostasis is accelerating. Two decades after the initial discovery in Drosophila that mutations in polo give rise to cells with aberrant mitotic spindles, we have achieved a relatively mature understanding of the biochemical role of polo kinases within cells [252]. This evidence is buttressed by the accelerated pursuit of molecular inhibitors of polo kinases for the treatment of human malignancy. Much of the knowledge about the polo family of kinases is the result of extensive investigation of homologs in model organisms. Drosophila polo is the homolog of Xenopus Plx1, human Polo- like kinase 1, fission yeast Plo1 and budding yeast Cdc5 [253-255]. Budding and 37 fission yeast have a single polo kinase, which most closely resembles Drosophila polo and human Plk1 in form and function; for this reason they are known collectively as polo kinases. In addition, metazoans have at minimum one additional PLK paralog (Plk4/SAK); human cells have two other family members Plk2 and Plk3 [256]. The polo kinase sequence and function is highly conserved throughout evolution [256]. Common among all polo kinases is the presence of an amino- terminal serine/threonine kinase domain and two distal polo boxes that fold into a functional polo-box domain (PBD) (Figure 5) [257, 258]. All of the known polo homologs and paralogs with the exception of Plk4/SAK, which has only a single polo-box, share this common structural fold [256]. The Plk1 PBD binds phosphorylated serine and threonine peptide sequences and demonstrates an extraordinary preference for serine and proline at the pThr -1 and +1 positions respectively [257-259]. Critical for phosphopeptide binding are conserved histidine and lysine residues (H538, K540 in Plk1) within the second polo-box that coordinate and neutralize the phosphate moiety. Additional residues including a conserved tryptophan (W414 in Plk1) within the first polo-box make significant contributions to phospho—peptide binding in Plk1 [257, 258]. In most organisms, including S. cerevisiae, increased protein expression of Polo kinase/Cch precedes catalytic activation of the kinase domain [260]. In the budding yeast Cdc5 levels are regulated predominately by activity of the th1-version of the APC [261]. Cdc5 protein levels are low throughout G1 when APC-th1 activity is high, but rise with increasing Cch8-Cln activity and peak in 38 late S-phase [260]. Protein levels remain stable throughout mitosis until activation of APC activity is fully restored following Cdc14 phosphatase release and mitotic exit [261]. In human cells regulation of Plk1 expression appears to be regulated by transcriptional repression in G1 and S-phase as opposed to transcriptional activation in late G2 [262]. The cell cycle-dependent element/cell cycle genes homology region (CDE/CHR) is immediately adjacent the Plk1 transcriptional start site and expression correlates negatively with CDE/CHR function [263]. In addition, the Rb family of pocket proteins (p107/p130) transcriptionally represses Plk1 by binding E2F4 directly on the promoter [264, 265]. Correct localization of Polo kinases within the cell is essential for proper regulation of cell cycle progression and growth. Experiments in vivo identified that Plk1 localizes to the mammalian centrosomes and kinetechores until the metaphase-to—anaphase transition {266-2691. At this stage of mitosis the affinity for these sites is diminished and Plk1 relocalizes to the spindle midzone in preparation for cytokinesis [266-269]. Similar to Plk1, Cdc5 localizes to the budding yeast spindle pole bodies and septin ring at the bud neck [94, 270]. In contrast to mammalian cells, Cdc5 targeting to SPBs occurs very early in S- phase and relocalizes to the bud neck prior to mitotic entry [271]. A functional Cdc5 PBD interacts with phosphorylated residues matching CDK consensus sites and is required for targeting Cch by binding to SPBs and the bud neck [257, 272]. Interestingly, bud neck localization appears dispensable for viability in budding yeast with only SPB localization being required for viability [114]. 39 Essential substrates involved in Cdc5 regulation of mitotic exit (discussed in more detail below) are located at the SPB and most likely explain this observation. Although the PBD is essential for targeting Cdc5 throughout the cell, the kinases that prime substrates for PBD are still largely unknown. Proteomic and structural analysis of optimal PBD binding sequences initially suggested that CDKs may prime proteins for Polo binding, however a recent study suggests that Plk1 binding can occur independent of substrate phosphorylation [244, 259, 273]. These studies suggest that our understanding of Polo targeting and regulation may be more complex than originally believed. GZIM Transition The key Cdk1 regulatory node for mitotic entry involves regulation by Plk1, however activation of Polo kinases and their relative importance in promoting mitotic entry varies considerably among organisms. This observed discrepancy might result from an inherent difference in the reliance of the 62 phase in monitoring cell cycle progression in divergent organisms. Vertebrates and fission yeast have a prolonged G2 phase and the G2/M checkpoint is a robust cell cycle arrest mechanism in the presence of DNA damage. Budding yeast by contrast have an extremely short 62 phase and rely more heavily on the spindle assembly checkpoint for delaying cell cycle progression. It may come as no surprise then that experiments in fission yeast, Xenopus and humans support a more prominent role for Polo kinases in acting upstream of Cdk1 [255, 274]. Plk1 phosphorylation and activation of the Cdc25 phosphatase at the G2/M transition is necessary for Cdk1 dephosphorylation and mitotic entry [255, 274]. Catalytic activation of Plk1 is regulated by cooperative action of the Plk1 interacting protein Bora and the Aurora A kinase [275]. Bora accumulation and binding to Plk1 in G2 phase relieves PBD autoinhibition of the catalytic domain and increases the accessibility of Aurora A to the Plk1 T-loop [257, 275]. This mechanism for Plk1 and subsequent Cdk1 activation is a critical target for cell cycle arrest and recovery in response to DNA damage [276]. Budding yeast experiments suggest a relatively minor role for Cch in regulating the G2/M transition. Published reports suggest that Cdc5 kinase activation is downstream of mitotic CDK, as catalytic activation of Cdc5 requires direct phosphorylation by Cch8, although the cyclin specificity for this interaction awaits elucidation [277]. In addition, cdc5-1 ts mutants progress through the G2lM transition normally and arrest in late mitosis with elongated spindles and partially divided nuclei [254]. In contrast to other organisms, Swe1 deletion has no measurable affect on growth or cell cycle progression in normal yeast [278]. It appears, however, that Swe1 plays a primary role in arresting cell cycle progression in response to activation of the morphogenetic checkpoint [279]. Chemicals that inhibit normal actin polymerization and bud formation prevent Hsl1-dependent localization of Swe1 to the bud neck [280, 281]. Once at the bud neck, Cdc5 and Cla4/PAK hyperphosphorylate Swe1, targeting it for ubiquitin- dependent proteosome degradation[282]. This degradation alleviates the antagonistic function of Swe1 on Cdk1 permitting mitotic entry. 41 Metaphase-to-Anaphase Transition Sister chromatid segregation occurs after activation of the Cdc20-version of the APC at the metaphase-to-anaphase transition [98]. Prior to chromosome separation, sister chromatids are held together by a family of proteins called cohesins (described above). Degradation of the Scc1 cohesin subunit is essential for chromatid resolution during anaphase and Polo kinases facilitate this loss in both mammals and yeast cells [283]. In vertebrates, phosphorylation of Scc1 and SA1/SA2 by Polo kinase reduces the affinity of cohesins towards chromatin resulting in the loss of cohesion along chromosome arms in prometaphase [284]. In budding yeast, the entire chromosome arms segregate simultaneously (with the exception of the rDNA locus and telomeric regions) following cleavage of Scc1 by Esp1/separase[92]. It is thought that phosphorylation of Scc1 by Cdc5 increases the affinity of Esp1 for Scc1, however this function of Cdc5 is dispensable and cells initiate anaphase without Cdc5 phosphorylation [275]. Regulation of Cytokinesis In addition to their role in mitotic exit, Cdc5, Cdc15 and Dbf2-Mob1 have additional roles in promoting cytokinesis in budding yeast. All three factors are required for proper actinomyosin ring formation at the budneck [285-288]. Following MEN activation and Cdc14 release, Cdc15 and Dbf2 relocalize from the SPB to the mother-daughter—budneck [119, 289, 290]. This is in contrast to Cdc5 localization, which targets to the budneck prior to mitosis where it participates in Swe1NVee1 downregulation at the GZ/M transition [114, 271, 282, 42 291]. cdc5 (ts) mutants demonstrate defects in septum closure and membrane fusion resulting in chained cell morphologies [291]. These experiments suggest that Cdc5 has cytokinetic functions separate from MEN activation, as some mutants remain competent for mitotic exit. In fission yeast, some members of the MEN, including Plo1, participate in the septation initiation network (SIN), which is required for cytokinesis [292, 293]. As in fission yeast, human Plk1 localizes to the spindle midzone and regulates contraction of the actin ring prior to abscission [267, 286]. Although the exact nature of cytokinetic regulation by Polo-like kinases awaits further elucidation, the degree of conservation between highly divergent organisms is suggestive of a critical role in regulating this process. CDC7-DBF4 AND PLK1 INVOLVEMENT IN CANCER ETIOLOGY The development and progression of human cancers requires genetic modification of multiple pathways including apoptotic regulation, cellular proliferation, migration and invasion. Pathways regulated by Cdc7-Ask and polo kinases put these proteins in a unique position to control and regulate cell growth in cancer cells. Deregulation of the cell cycle is a hallmark of human cancer and the discovery that normal Cdc7 and polo kinase activity is perturbed in some tumors has important implications for the biology and treatment of these diseases. Polo-like Kinases 43 Elevated Plk1 activity contributes to many of the phenotypes (cellular proliferation and genome instability) that are frequently associated with malignant disease. Indeed, overexpression of Plk1 in NIH 3T3 cells induces colony formation in soft agar and tumors in nude mice, suggesting a causative role in driving malignant progression [294]. It is not surprising, therefore, that deregulation of polo kinases is found in many human cancers. Transcriptional expression of Plk1 has been extensively studied in myriad . cancer types and tissues. Plk1 expression is elevated in breast, head and neck, squamous esophageal, ovarian, gastric, colon, endometrial carcinoma and glioblastomas [295]. In addition, increased expression is positively correlated with poor prognosis in many cancer types. In non-small cell lung adenocarcinomas, patients expressing moderate Plk1 levels demonstrated a significantly better prognosis (5-year survival mean; 52%) than patients expressing high levels of Plk1 (5-year survival mean; 24%) [296]. Similar results were observed in squamous cell esophageal carcinomas with low level expressers demonstrating a significant increase in 3-year survival (3-year survival mean; 54%) over high expressers (3-year survival mean; 25%) [297]. In large part, immunohistochemical analysis of Plk1 protein expression in solid tumors has supported these findings. In ovarian carcinoma patients, the percentage of Plk1 cells was positively associated with tumor grade, with more aggressive grades demonstrating more robust Plk1 expression (percent positive cells: grade 1, 13.5%; grade 2, 21.9%; grade 3, 35%) [298]. Similar findings were reported in glioma patients; tumors demonstrating more advanced anaplasia had significantly higher levels of Plk1 expression compared to low-grade and “normal” tissues [299]. In addition, Plk was positively correlated (r= 0.519) with MlB-1 expression in these cells. Interestingly, Plk1 was also found to positively correlate (r = 0.677) with estrogen receptor (ER) expression in human breast carcinoma cells [296]. Although the biochemical significance of this observation awaits further elucidation, the relationship between these two proteins is interesting considering the strong association with ER status and patient prognosis. Among polo kinase family members, Plk3 has also received significant attention with regard to its involvement in regulating cancer progression. In contrast to Plk1, however, a significant percentage of studies suggest that Plk3 expression is positively associated with patient survival. Like Plk1, the expression of Plk3 is regulated throughout the cell cycle and peaks in late S and M phase [300]. Plk3 mRNA and protein are undetectable in lung cancers and is similarly reduced in human head and neck carcinomas and rat colon tumors [300-302]. In support of these findings, induction of Plk3 reduces fibroblast proliferation and induces cell cycle arrest and apoptosis [303, 304]. Therapeutics Targeting Mitotic Kinases (Polo and Aurora kinases) Cells undergoing mitosis are particularly susceptible to apoptotic-induced cell death [305]. For this reason, many of the most historically successful anti- neoplastic compounds target this stage of the cell cycle. In particular the vinca alkaloids and taxanes, which disrupt normal microtubule dynamics, perturb mitotic spindle mechanics resulting in activation of the mitotic checkpoint, cell 45 cycle arrest and apoptosis [306]. Absence of cancer cell specificity, however, results in deleterious off-target side effects and significant peripheral neuropathies in most patients. As a result, pharmaceutical companies have invested significantly in the development of compounds targeting specific mitotic kinases: most notably the Aurora kinases and Plk1. Aurora kinases in particular have received increasing interest over the last several years because of their requirement for chromosome alignment and partitioning during mitosis [307]. Inhibition of Aurora A kinase activity generates cells with monopolar spindles that arrest at the SAC and eventually undergo apoptosis [308]. Aurora B inhibition by contrast results in SAC bypass and mitotic progression despite a failure to segregate chromosomes. This results in aneuploidy and tetraploidization of cells and ultimately apoptosis [309]. Specific (AZD1152, GSK1070916 and MLN8237) and pan-aurora kinase (CYC-116) inhibitors have been developed and are in Phase I and II clinical trials [310-312]. In preclinical studies, compounds showing marked anti-tumor activity, regardless of the pharmacokinetic profile, suggested that Aurora A and Aurora B are both effective targets for therapeutic intervention in cancer cells. Currently, two strategies exist for targeting Plk1 in cancer cells. Firstly, inhibition of the Plk1 kinase domain has resulted in the development of numerous small ATP-binding pocket inhibitors (Bl 2536, GSK461364, HMN-214 and ON 01910) [313-318]. These compounds are currently being tested in Phase I and II clinical trials and their effectiveness awaits publication. Secondly, peptides that target the Plk1 polobox domain were shown to decrease the kinase activity. Development of the first non-peptide PBD inhibitor Poloxin was shown to induce mitotic arrest and apoptosis in HeLa cells [319, 320]. It might be expected that as our ability to more accurately design inhibitors targeting cell cycle kinases improves more promising results may emerge from clinical trials regarding cell cycle targeted therapies. Cdc7-Dbf4 Cancer Involvement Compared to Plk1, our current knowledge of the role Cdc7-Dbf4 plays in cancer development and progression is relatively modest, although recent studies indicate a possible role for DDK in driving malignant progression. Overexpression of CDC7 mRNA in human tumors and cell lines first suggested a role for DDK in regulating cancer cell proliferation [321]. This observation is supported by further studies demonstrating increased Cdc7 and Dbf4 protein levels in a wide variety of human tumors and cell lines [322]. In support of this finding, Dbf4 expression levels negatively correlate with patient survival and tumor relapse in cutaneous melanoma cells. Consistent with this finding, knockdown of Cdc7 expression in cells induces apoptosis [322-324]. Interestingly, overexpression of Cdc7 and Dbf4 arrests normal chinese hamster ovary cells at the G2/M transition, suggesting that inactivation of specific checkpoints may be a precondition for increased Cdc7-Dbf4 activity [325]. Another hallmark of tumor development is that most malignant cells are genetically unstable and contain a high degree of DNA damage. As a 47 consequence, tumor cells develop mechanisms to bypass cell cycle checkpoints in response to damaged DNA. The tumor suppressor p53 is a key target of the DNA damage checkpoint in mammalian cells and transcriptionally activates key proteins involved in cell cycle arrest and apoptosis [326]. Depletion of Cdc7 also suggests a p53-dependent replication checkpoint in cancer cells. Some cells that are depleted of Cdc7 are susceptible to DNA damage during replication and require p53 to maintain cell viability and avoid apoptosis [324, 327]. A link between p53 and Cdc7 expression in maintaining cell viability in stressed cells is further supported by work in our lab demonstrating a correlation between p53 loss and increased Cdc7-Dbf4 expression [322]. These studies suggest functional interplay between Cdc7-Dbf4 and p53 in maintaining cancer cell viability in the presence of genome stress. As a result, these reports have important implications for the development of Cdc7 kinase inhibitors as a possible therapeutic drug target in p53 mutant tumors. Recent reports of novel Cdc7 kinase inhibitors report inhibition of cellular malignant growth in vitro and in xenograft mouse models [328-330]. Clearly, our understanding of the possible role Cdc7 plays in tumor development and/or progression is still in its infancy and further experimentation is required to investigate whether any serious effort at targeting Cdc7 in human tumors would be efficacious. Significance and Concluding Remarks Elucidating the mechanism of DNA replication and initiation during S- phase is a fundamental problem in eukaryotic cell biology. A well-established 48 causal link between DNA replication damage and cancer initiation suggests a thorough understanding of this process has the potential to improve human health. Critical to a more mature understanding of replication in eukaryotes is further elucidation of the role DDK (Cdc7-Dbf4) plays in regulating this process. In addition to the general involvement of replication and DNA damage repair defects in driving malignant progression, emerging evidence suggests direct involvement of human Cdc7-Dbf4 in cancer progression and/or maintenance and highlights the importance of additional investigation into DDK function. Dbf4 structure/function studies identified and characterized three regions important for Cdc7-Dbf4 function [227, 229, 231]. As discussed, budding yeast DBF4 encodes three regions (N, M & C) of high interspecies sequence similarity [227]. Although characterization of the M and C regions in Cdc7 binding and activation has improved our understanding of Dbf4, the biochemical function of the Dbf4 N-terminus remains enigmatic. The discovery that mutations within Dbf4 region N confer sensitivity to DNA damaging agents and eliminate Rad53- dependent checkpoint phosphorylation on Dbf4 raise the possibility that the Dbf4 N-terminus may regulate cellular responses to DNA damage. The well- established role for Chk2 (yeast Rad53 homolog) involvement in human cancers and identification of somatic mutations of Chk2 in human breast cancer patients suggests this research could have important implications for our understanding of cell biology and human disease. Critical to understanding this process is the identification of additional proteins that interact with the N-terminal region of Dbf4. Masai and colleagues 49 reported that fission yeast DDK (Hsk1-Dfp1) interacts with components of stalled replication forks, but it was not clear whether this interaction was dependent on the Dbf4 N-terminus or conserved in the budding yeast [238]. To identify the function of the Dbf4 BRCT domain in these processes, our lab performed a yeast two-hybrid screen and identified an interaction between the N-terminus of Dbf4 and Cdc5. An interaction between Dbf4 and Cdc5 was initially reported in 1996, however this study did not determine the biochemical mechanism of the interaction and proposed a role for Cdc5 function in replication [331]. Subsequent studies failed to support this hypothesis on the primary basis that Cdc5 expression and kinase activity are not activated until late S or early G2 phase [260]. Additionally, evidence of a role for Cdc5 in the regulation of Dbf4 during DNA replication is unproven. As a result of emerging evidence implicating members of the Cdc5 family of Polo-like kinases in positively regulating the G2/M transition, events in anaphase, mitotic exit and cytokinesis, we hypothesized that Dbf4 may regulate Cdc5 to prevent premature activation of events in mitosis before completion of DNA replication [112, 115, 232, 233, 270, 271, 332]. We reasoned that this finding would represent a significant advance in our understanding of Cdc7-Dbf4 biology as it would be the first report of a function for Dbf4 outside S-phase. In addition, the central role for Dbf4 and Cdc5 in regulating replication and mitosis, respectively, their involvement in promoting cancer progression and recent interest in targeting both kinases therapeutically in human cancers underscored the importance for additional investigation into the functional regulation of these 50 proteins. 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Cdc7p-dbf4p regulates mitotic exit by inhibiting Polo kinase. PLoS Genet 5, e1000498. 81 ABSTRACT: Cdc7p-Dbf4p is a conserved protein kinase required for the initiation of DNA replication. The Dbf4p regulatory subunit binds Cdc7p and is essential for Cdc7p kinase activation, however the N-terminal third of Dbf4p is dispensable for its essential replication activities. Here we define a short N-terrninal Dbf4p region that targets Cdc7p-Dbf4p kinase to Cchp, the single Polo kinase in budding yeast that regulates mitotic progression and cytokinesis. Dbf4p mediates an interaction with the Polo substrate-binding domain to inhibit its essential role during mitosis. Although Dbf4p 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, Cdc7p-Dbf4p prevents inappropriate exit from mitosis by inhibiting Polo kinase and functions in the spindle position checkpoint. INTRODUCTION: Accurate ordering of cell cycle events is an important requirement for the viability of all eukaryotic organisms. Once cells commit to duplicate their genome they must restrain mitosis until replication is complete and then accurately coordinate mitosis with cytokinesis to ensure the faithful transmission of chromosomes to daughter cells [1]. lmportantly, errors in cell cycle checkpoints that enforce this ordering can be deleterious for accurate chromosome transmission. For 82 instance, DNA damage or replication fork arrest during S-phase elicits a reversible block to mitotic progression by the budding yeast Mec1 p (HsATR) and Rad53p (HsChk2) checkpoint kinases [2, 3]. In the absence of Mec1p or Rad53p, replication fork arrest during S-phase is not sensed leading to premature mitotic events and cell death (reviewed by [4]). Additionally, since daughter cell growth is highly polarized in the budding yeast, exit from mitosis is prevented until sister chromatids segregate through the bud neck and into the daughter cell [5-7]. This ensures that spindle disassembly and mitotic exit are not initiated until accurate chromosome partitioning between mother and daughter cells has occurred. Failure to block mitotic exit when nuclear division takes place within the mother cell results in polyploid and anucleate progeny [8, 9]. It is not surprising therefore, that both entry into and exit from mitosis are delayed by cellular checkpoints that respond to replication stress, chromosome damage, or spindle disruption [1]. Errors in these mitotic checkpoints are catastrophic and result in ploidy defects and genetic alterations, which are frequently observed in human cancers (reviewed by [10]). The Cdc7p-Dbf4p kinase is required to catalyze the initiation of DNA synthesis at the beginning of S-phase (reviewed by [11]). Cdc7p kinase activity is tightly regulated during the cell cycle by binding the Dbf4p regulatory subunit, which is cyclically expressed. Dbf4p accumulates in late G1, is present throughout S-phase and then is destroyed during mitosis and early G1 by anaphase promoting complex (APC)-dependent degradation [12-17]. Therefore, Cdc7p-Dbf4p kinase activity is low following exit from mitosis and entry into G1- 83 phase until it is needed to initiate a new round of DNA synthesis in late G1-phase of the following cell cycle. Multiple lines of evidence suggest that Cdc7p-Dbf4p activates the MCM DNA helicase [18-20] that is assembled at origins of replication in early G1 in an inactive form (reviewed in [21, 22]). In addition to its essential role in replication initiation, several studies suggest that the Cdc7p-Dbf4p kinase responds to DNA damage or replication fork stalling but its precise role in these activities is unknown [17, 23-25]. Dbf4p encodes a dispensable BRCT-like domain in the N-terminus that might target the kinase to stalled replication forks [26, 27]. In fission yeast, the Cdc7p-Dbf4p ortholog Hsk1p-Dfp1p interacts with Swi1 p (budding yeast Tof1 p), a component of replication forks required for fork stability and also promotes centromeric cohesion in early mitosis [28, 29]. Rad53p also phosphorylates Dbf4p in response to replication stress and this regulation requires N-terminai Dbf4p sequences through which Rad53p physically interacts [17, 25, 30]. interestingly, the absence of the BRCT-like domain results in a defect in late origin activation suggesting that this domain might alter Cdc7p-Dbf4p binding at early versus late replication origins [26]. Together, these data suggest that the Dbf4p N-terminus encodes non-essential regulatory functions that target the kinase to particular substrates. To identify proteins that interact with the Dbf4p N-terrninus, we performed a yeast two-hybrid screen with an N-terminal region of Dbf4p and identified an interaction with the Cdc5p kinase, the only Polo ortholog in yeast. Budding yeast Polo, like Drosophila Polo and human Polo-like kinase 1 (Plk1), functions as a 84 master regulator of mitotic progression and is also required for cytokinesis (reviewed by [31, 32]). Polo activity is regulated by several independent cellular mechanisms. Polo protein levels are controlled by APC-dependent degradation in mitosis/G1-phase and activation of Polo catalytic activity requires phosphorylation by Cdk1 kinase early in G2 [33-35]. In addition, Polo function is inhibited by cell cycle checkpoints that are induced following DNA or spindle damage [36-38]. A genetic and physical interaction between Dbf4 and Polo was described previously [39, 40], however the biological significance of this interaction was not known. Polo controls multiple mitotic events to ensure accurate chromosome segregation. After anaphase initiation, Polo is required to activate the FEAR (Cdc14 early anaphase release) and MEN (mitotic exit network) pathways that promote nucleolar release of Cdc14p phosphatase [37, 4143]. Limited Cdc14p release by the FEAR pathway promotes accurate rDNA and telomere segregation [4446]. Subsequent full nucleolar release of Cdc14p by the MEN reverses Cdk substrate phosphorylation that leads to APC-th1 p activation, cyclin destruction and mitotic spindle disassembly (reviewed by [47]). Activation of the MEN is promoted by Tem1p-GTP and antagonized by Bfa1p-Bub2p, a two-component GTPase activating protein (GAP) [48-50]. To promote mitotic exit, Polo phosphorylates Bfa1p-Bub2p to inhibit its GAP activity and is also required for activation of Dbf2p kinase activity, independently of Bfa1p-Bub2p [37, 49, 51, 52]. The Polo requirement for Dbf2p kinase activation may reflect that Polo also promotes Cdc14p release in the FEAR pathway, which primes the 85 MEN [43]. Therefore, Polo promotes accumulation of Tem1p—GTP and activation of the downstream MEN kinases Cdc15p and Dbf2p, which ultimately cause full release of Cdc14p from the nucleolus. In response to replication fork arrest, Rad53 inhibits MEN activation, which may or may not impact Polo activity since the molecular basis of this regulation is not understood [53, 54]. Spindle position defects also counteract Polo activity by targeting Kin4p kinase to the spindle poles where it inhibits Polo-dependent Bfa1p phosphorylation [8, 9, 55]. Failure to execute the spindle position checkpoint (SPOC) results in premature exit from mitosis and nuclear partitioning defects. Here we define an N-terrninal Dbf4p golo-box jnteraction region (that we refer to as the “PIR”) that binds directly to Polo and show that Dbf4p inhibits Polo and Dbf2p activity. Deletion of the PIR allows Cdc14p nucleolar release in a cdc5-1 mutant at the non-permissive temperature. In response to nuclear mispositioning, a dbf4 mutant lacking the PIR fails to arrest in mitosis and prematurely exits the cell cycle. Thus, Dbf4 protein is required for proper functioning of the spindle position checkpoint most likely by antagonizing the ability of Polo to promote Cdc14p release in either the FEAR or MEN pathways. Our work therefore reveals a previously unrecognized function for Dbf4p in the regulation of mitotic progression through a direct interaction with Polo. RESULTS: The Cdc5p polo-box domain interacts with the Dbf4p N-terminus. We conducted a yeast two-hybrid screen to identify proteins that interact with the 86 Dbf4p N-terminus (residues 67—227) and recovered multiple clones encoding the polo-box domain (PBD) of Cchp. Polo kinase has two conserved domains; an N-tenninal kinase domain and a C-terrninal region called the polo-box domain (PBD) (reviewed by [56]), which is a phospho-Ser/T hr binding module that targets the kinase to its mitotic substrates [57, 58]. The crystallographic structure of the Plk1 PBD bound to a phospho—threonine peptide has been solved [59]. Since the Dbf4p BRCT-like region alone (residues 110-227) failed to interact with the Polo PBD (Figure 6A), this suggested that the PBD interaction was occurring through Dbf4p N-tenninal sequences from 67-109. Residues 67-109 were similarly required for the Polo interaction within the context of full length Dbf4p (Figure 6B) and were sufficient to interact with the Polo PBD (Figure 6A). Dbf4p residues 67-109 with all serines/threonines changed to alanine still interacted with the PBD (Figure 6A) suggesting that the PBD can bind to this Dbf4p region independently of phosphorylation. Further deletion and point mutant analysis (Y- C.C. and MW, unpublished data) revealed that residues 82-88 are essential for the Dbf4p-Polo interaction (Figure 6A, B). The PBD is composed of three conserved regions called the Polo-cap (Pc), Polo-box 1 (P31) and Polo-box 2 (P82) that fold together to form a functional phosphopeptide-binding domain [59]. We deleted conserved residues within the PBD to test their requirement for interaction with Dbf4p (Figure 60). Deletion of residues preceding the PBD (GAD-Polo 454-705) had little effect on the Dbf4p-Polo interaction. However, elimination of the Po (GAD-Polo 510-705) 87 Figure 6. The N-terminus of Dbf4p interacts with the Polo PBD. Cells co- transformed with various bait _G_Al.4 DNA Qinding gomain fusions (GBD-DBF4) and prey QAL4 activation gomain fusions (GAD-0005) constructs were spotted at 10-fold serial dilutions on SCM—Trp -Leu and SCM—Trp—Leu—His plates containing 2mM 3AT (A, C) or 0.5mM 3AT (B) and incubated at 30 degrees Celsius for 2 to 3 days. The GBD-Dbf4 protein levels are shown in Figure 7. 88 DmnTmooo Dmn.-mouo HloLn. £1- :3- 9...- c0_uom._0uc_ IN :m... n..... .m. ..I. J 0 m=mo _m~o._. <<<< mow I. me me _.1 hm mw-Nw< “5mm M \ NNN no Rwfllo: R031 5 Louoo> :m 89 m..-.- so..- 9 ...- :o-.- 9 ...- woulllllowm mon:|l|l||.H.I¢mv alum monllllfllwmv nomm Nun. Em on. 93> . 9n. mono 5.899... In «.60 .80... ..mm #30 o m....- :3- 9 ...- :o-_- 9... - Lemma «New emu-mono o o o voslnHullI-fllfll S n.. o o sTnUanILflul. 88> 37;. m 0 EOE .2 EOE momm 8892:. IN £8 53 .E00 .. 2:9". completely disrupted the interaction with Dbf4p. These data suggest that the structural integrity of the Polo PBD is required for Dbf4p binding. Dbf4p directly interacts with Polo. Dbf4p binds and activates the Cdc7p kinase subunit in yeast and has no known role apart from its interaction with Cdc7p [14, 60]. To determine whether the interaction between Dbf4p and Polo occurred in the context of the full-length Cdc7p-Dbf4p kinase, Sf9 cells were co-infected with baculoviruses expressing Polo, wild type HA—Cdc7p-Dbf4p or wild type HA- Cdc7p with various Dbf4p deletion derivatives. HA-Cdc7p-Dbf4p kinase was immunoprecipitated using an antibody against the HA tag and examined for the presence of Polo. All the Dbf4p deletion derivatives we examined interact with Cdc7p and activate normal Cdc7p kinase activity ([26] and data not shown). Whereas Polo interacted with full-length Cdc7p-Dbf4p and Cdc7p-Dbf4-NA65p, Polo did not interact with Cdc7p-Dbf4p complexes that lacked the Dbf4p N- terminal 109 residues required for the Polo two-hybrid interaction (Figure 8A). These data indicate that full length Cdc7p-Dbf4p kinase interacts with Polo but that Dbf4p residues 65—109 are required for this interaction. lmportantly, HA- Cdc7p-Dbf4p interacts with Cdc5p in yeast when the proteins are expressed at endogenous levels and this interaction also depends on the Dbf4p N-terminus (Figure 88). We next tested whether Polo bound directly to Dbf4p using purified proteins. GST-PBD and Sumo-Dbf4 67-109 fusion proteins purified from E. coli were mixed, pulled down using glutathione-Sepharose and analyzed by 91 Q g °9 L 3% R988 31s Est-:5; Egeé sees gags 0.0.0.0. 0.0.0.0. oooo oooo mmmm comma: 0000‘ c9000 a—MYC ..-.- --.—fl PonceauS Figure 7. Western blot of Gal4—DNA binding domain fusions to various Dbf4 N- terminal fragments for the 2-hybrid assays shown in Figure 6. immunoblotting. Although Sumo alone did not interact with GST-PBD, Sumo- Dbf4p 67-109 interacted with GST-PBD but not with GST alone (Figure 8C). These data indicate that Dbf4p residues 67-109 (that we refer to as the Dbf4p PIR) are sufficient for a direct interaction with the Polo PBD. Dbf4p inhibits Polo activity. Cdc7p-Dbf4p is required to initiate DNA replication, but is present throughout S-phase and during the metaphase to anaphase transition. Dbf4p is subject to APC-Cdc20p dependent degradation [16] but some protein is still present in late mitotic mutants that have activated the Cdc20p but not the th1p form of the APC [17]. We examined Dbf4 protein abundance relative to Pds1 protein in cells moving synchronously through the cell cycle, since Pds1 p is degraded at the onset of anaphase by APC-Cdc20 [61]. We found that although the abundance of both proteins declines at the same time, Pds1 p is absent during mitosis while some fraction of Dbf4p persists (Figure 9). In contrast, Dbf4p has very low abundance or is absent in cells arrested in G1-phase by mating-pheromone when the APC-th1p is active [12, 13, 15, 17] and Dbf4p is stabilized by inactivation of the APC in G1 or by removal of its N-terrninal D-box [13, 15-17]. Together these data suggest that Dbf4p degradation can occur via both APC-Cdc20p and APC-th1 p mediated ubiquitylation. ' Since budding yeast Polo is not required for DNA replication but promotes multiple mitotic activities, we reasoned that Cdc7p-Dbf4p might influence Polo activity during mitosis. The dbf4-NA109 mutant progresses normally through the 93 Figure 8. Dbf4p residues 67-109 interact directly with the Polo PBD. (A) HA- Cdc7p Dbf4p kinase or HA-Cdc7p plus Dbf4p truncation mutants were expressed in Sf9 cells together with Polo and immunoprecipitated using 120A5 antibody. Blots were probed with anti-Myc (Cdc5p), anti-Cdc7p and anti-Dbf4p polyclonal antibodies. (B) Endogenous HA3-Cdc7p-Dbf4p complexes were immunoprecipitated using 120A5 antibody from HA3-CDC7 DBF4 CDC5-Myc15 (M2741) and HA3-CDC7 dbf4-NA109 CDC5-Myc15 (M2743) yeast strains following nocodazole arrest and probed for Cdc7, Dbf4, and Cdc5-Myc. (C) Purified Sumo and Sumo-Dbf4 67-109 proteins were co-incubated with purified GST-PBD or GST alone; proteins were pulled down using glutathione-Sepharose beads and blotted with antibodies against GST or Sumo. (D) In vitro phosphorylation of GST-PBD using purified Cdc7p-Dbf4p kinase. 94 Geno. 022-8 mono-8 n: ...-m; -880 .88. "52.5 $8.5 -_ 1|..-" -anooo - n.. . II keno-8 1..-- . ; - - 1 . Ego-s mo; , , - £80 1 - £80 -.-.I . ., .. - ce..—an. n..-- ++++II ++++IINOUO- 100 CD 8o 95 Rebudding N 4: o o O 0 60 120 180 240 minutes after release from a-factor 120 —0— wild type + dbf4-NA109 + Abub2 —e— Abub2 dbf4-NA 109 8. 22-2% «82 «.88 8. 22-2% «82 «.88 «82 «.88 «82 «.88 8. 22-2% «.88 8. 22-2% «.88 «-88 , «82 m 0.50 m. 9.8.". 121 defects but deletion of both genes is lethal. In response to nuclear misorientation, S. cerevisiae inhibits premature activation of the MEN via the spindle position checkpoint (SPOC). When the SPOC is activated the Kin4p kinase localizes to spindle pole bodies (SPB) to counteract Polo Bfa1p phosphorylation [8, 9, 55]. Kin4p thus counteracts Polo inactivation of the Bfap1-Bub2p GAP and this inhibits MEN activation. Failure to adequately respond to nuclear mispositioning allows inappropriate nuclear division within the mother cell, leading to anueploidy and loss of viability. Given that Polo Bfa1p phosphorylation is prevented when the SPOC is activated, we hypothesized Dbf4p may also inhibit Polo to prevent mitotic exit in response to nuclear misorientation. To examine whether Dbf4 regulated cell growth in dyn1 and kar9 mutants in the presence of spindle poisons, strains were spotted at serial dilutions on media containing the spindle poison benomyl. We analyzed wild type, dbf4- NA109, kar9A, kar9A dbf4-NM 09, dyn1A and dyn1A dbf4-NA109 for growth. Deletion of KAR9 or Dyn1 alone negatively affected growth on benomyl as compared to wild type; interestingly, PIR deletion further decreased cell viability in both genetic backgrounds suggesting a role for Dbf4 in regulating cell viability in the presence of a perturbed spindle (Figure 17). 122 10° ' --0- wild type 90 - +Abub2 80 . +Acdc55 '8' Acdc55 dbf4-NA109 u 70 - +Akin4 dbf4-NA109 .E -o— Akin4 Acdc55 dbf4-NA109 'u 60 - '3 8 50 "' a: 40 - 39 30 - 20 - 10 - --- _. .- 0 50 100 150 200 Minutes after release from on factor Figure 16. Deletion of the Dbf4 PIR does not affect rebudding in the presence of spindle poison. WIId type, bubZA, cdc55A, cdc55A dbf4-NM 09, kin4A dbf4- NA109 and kin4A cdc55A dbf4-NA109 cells were synchronized with alpha-factor and released into YPD containing 15ug/ml of nocodazole at 30 degrees Celsius. Samples were quantitated for the percentage of rebudded cells (large budded cells with a new bud). 123 12.5 uglml ben 15 uglml ben wild type dbf4-NA109 kar9A kar9A dbf4-NA109 kar9A dbf4-NA 1 09 dyn 1A dyn1A dbf4-NA109 dyn1A dbf4-NA109 130...... Figure 17. PIR deletion decreases cell viability in the presence of spindle poisons. Strains were spotted at 10-fold serial dilutions on YPD with and without the spindle poison benomyl. Cells were grown at 30 degrees Celsius for 3 days. 124 To test more specifically whether DBF4 inhibited mitotic exit when nuclei were mispositioned, we examined wild type, dbf4-NA109, kar9A and kar9A dbf4- NA 109 strains for evidence of mitotic exit in the presence of mispositioned nuclei. Asynchronous cultures were grown at 25 degrees Celsius and shifted to 30 degrees Celsius for 4 or 24 hours prior to analysis to increase the penetrance of the nuclear mispositioning phenotype. Although wild type and dbf4 mutant cells did not misorient their nuclei (Figure 18), both kar9A and kar9A dbf4-NA109 strains had approximately equal number of cells with nuclear positioning defects- Importantly, deletion of the Dbf4p PIR resulted in a 2 to 3-fold increase (to 6%) in binucleate, anucleate and multinucleate cells at 4 hours (Figure 18A, 0, D), which was CDC5-dependent, suggesting that premature mitotic exit occurred. At 24 hours the number of aberrant chromosome segregation events due to loss of the Dbf4p PIR increased six-fold (12%) relative to kar9A alone (2%). Since exit from mitosis causes spindle disassembly, we quantitated the spindle morphology in cells containing correctly segregated nuclei (between the mother and daughter cells) and in those cells where anaphase had initiated solely within the mother cell. Although kar9A and kar9A dbf4—NM 09 cells had similar frequencies of intact or disassembled spindles when nuclear division proceeded normally, kar9A dbf4-NA109 cells showed a three-fold increase in spindle disassembly within the mother cell (leading to a bi-nucleate mother cell) compared to kar9A single mutants at 4 hours (Figure 18D). These data indicate that mitotic exit occurs in these cells in the absence of the Dbf4p PIR. 125 Figure 18. Deletion of the DBF4 PIR allows exit from mitosis in cells with misoriented nuclei. (A, B) Representative cells showing nuclear positioning and mitotic arrest defects. Nuclear position was analyzed after DAPI staining. Cells with anaphase spindles located exclusively in the mother cell body are noted (white arrow). Anucleate (*), binucleate (**) and multinucleate (***) cells were frequently observed in kar9A dbf4-NA109 (A) and dyn1A dbf4-NA109 (B) cells. (C, E) Quantitation of nuclear segregation defects. >300 cells were counted for each strain and nuclear position was represented as a percentage of the total cells in culture. Black bars represent cells with nuclei segregated between mother and daughter cells; gray bars, divided nuclei in the mother cell; white bars, multinucleate and anucleate cells. (D, F) Spindle morphology of strains with misoriented nuclei and spindles. >50 cells were counted for each genotype. Black bars represent cells with mitotic spindles. Gray bars represent cells that have exited mitosis. 126 Akar9 dbf4-NA109 DNA Tubilin M “ Adyn 1 Briiht Ad yn 1 dbf4-NA 109 Figure 18 Cont. 4 hours C 16 .8796 12 I ®@ 2 [:00 3 .- i 32 009:9 ‘ h M 0 WT NA109 Akar9 Akar9 Akaro NA109 ~4109 odes-1 12 24 hours fl 8 T) o 32 4 0 WT NA109 Akar9 “W9 NA109 D 80 2 360 3240 20 Akar9 Akar9 4"“ ~4109 “3’9 ~4109 I96 I®O S©© 3630 E Figure 18 Cont. I68 IGDO WT ”mos A 1 Ab b2 My“ Ady’" dyn " NA109 Abub2 16 24 hours % Cells a: WT NA109 Ad 1 Ab b2 AM’ “'7'" y" " NA109 Abubz F 100 80 60 40 20 % Cells 0 n1 Adyn1 Adyn1 Ad n1 Adyn1 Adyn1 Ad” NA109 Abub2 y NA109 Abub2 Im I09 I®OI®O 129 Similarly, we tested whether deletion of the Dbf4p PIR allowed premature mitotic exit in cells disrupted in the dynein pathway (Figure 18B, E, F). The spindle-positioning defect associated with deletion of DYN1 is especially prominent at low temperatures. Although after 8 hours at 14 degrees Celsius only ~3% of dyn1A cells had exited mitosis inappropriately, deletion of the Dbf4p PIR resulted in a 3- to 4-fold increase in mitotic exit as evidence by the appearance of multinucleate and anucleate cells (Figure 18E) and this frequency was increased at 24 hours. For comparison, a dyn1A bubZA strain had a similar but higher frequency of segregation defects (Figure 18E, F). Therefore, the bypass of the SPOC following loss of the Dbf4p-Polo interaction is comparable to deletion of the MEN inhibitor BUBZ. As with the kar9A mutant, we observed that a higher percentage of dyn 1A dbf4-NA109 cells compared to dyn1A single mutants that divided nuclei within the mother cell had disassembled their spindles (Figure 18F). These observations indicate that deletion of the Dbf4p N-terminus (including the PIR) overrides the mitotic arrest normally activated by the SPOC. DISCUSSION: We found that Dbf4p inhibited Polo kinase during mitosis very likely through a direct interaction with the polo-box domain. This interaction inhibited MEN pathway activation, nucleolar release of the Cdc14p phosphatase and was likely critical to maintain genome integrity during activation of the spindle position checkpoint. These results therefore have important implications for 130 understanding Dbf4p function and the regulation of mitotic progression in eukaryotic cells. Defining the interaction between Dbf4p and Polo. The N-terminal third of Dbf4p encodes multiple functions: a destruction box (residues 62-70), two putative nuclear localization signals (residues 55-61, 251-257) and a BRCT-like domain (residues ~117-220). Nonetheless the 265 N-terminal amino acids of Dbf4p are not essential as long as a nuclear localization signal is present [26]. Deletion of the Dbf4p N-terminus through the PIR has no observable effect on growth, viability, or cell cycle progression either under normal growth conditions or in the presence of replication or spindle poisons ([26] and data not shown). Here, we discovered an interaction between Dbf4p and the Polo PBD that mapped to a short sequence of ~40 amino acids preceding the BRCT-like domain. Although a two-hybrid interaction between Polo and Dbf4p was reported before [39], the significance of this interaction was not determined. The Polo PBD functions as a module for binding phosphorylated proteins and thereby targets Polo to its cellular substrates [57, 59]. The question naturally arises as to whether phosphorylation of the Dbf4p PIR is required for PBD binding. Currently, our observations suggest that phosphorylation is not required. A polo-box binding consensus sequence (S(pS/pT)P/X) is not present within this region of Dbf4p [59] and mutation of all putative serine and threonine residues within the Dbf4p PIR did not significantly diminish the interaction in the two-hybrid assay. These data suggest that phosphorylation of Dbf4p is not crucial for Polo 131 binding. Our observation that the Dbf4p PIR purified from E. coli directly interacted with the Polo PBD also supports this notion. Thus phosphorylation of Dbf4p was not critical for binding to Polo in vitro, but we cannot exclude the possibility that phosphorylation contributes to Dbf4p-Polo binding in vivo when the two proteins are present at physiological concentrations. When does Dbf4p inhibit Polo and mitotic exit? The finding that deletion of the Dbf4p PIR significantly suppressed the ts phenotype of cdc5-1 suggests that Cdc7p-Dbf4p inhibits Polo during the normal cell cycle and perhaps during periods of replication stress, when Cdc7p-Dbf4p is stabilized [17]. Our data clearly demonstrate a role for Dbf4p in inhibiting mitotic exit, since loss of the Dbf4p PIR suppressed both cch and dbf2 ts mutants and allowed sustained Cdc14p phosphatase release and cytokinesis in the cdc5-1 mutant at the NPT. However, during an unperturbed cell cycle the absence of this regulation had little impact. This is likely attributable to the fact that the cell cycle regulation of Polo activity is complex and modulated by multiple cell cycle checkpoints. Since dbf4-NA109 bubZA double mutants were more sensitive to growth on spindle poisons than either mutant alone (Figure 19), the Dbf4p-Polo and Bfa1p-Bub2p pathways may work together to suppress premature activation of the mitotic exit network. It was shown very recently that increased expression of a non-destructible form of Dbf4p (Dbf4-N465p) could delay rDNA segregation when Clb5p was also stabilized [16]. This raises the possibility that Dbf4p inhibits Cdc14p release via 132 YPD wildtype . . Q a .. dbf4-NA109 O O o e Amad2 . O 0 a3 Amad2 dbf4-NA109 . O C :9 Abub2 . O O Abub2 dbf4-NA109 . O Figure 19. Deletion of the Dbf4p PIR causes decreased growth in the presence of spindle poisons. Indicated strains were spotted at 10—fold serial dilutions on YPD or YPD containing 10pg/ml benomyl. 133 the FEAR pathway under some circumstances and is consistent with our data showing that Dbf4p is a Polo inhibitor. However, we found no evidence that the dbf4-NA109 allele promoted premature rDNA segregation (a FEAR pathway event) in the cdc5-1 mutant (Figure 20A). Similarly, we found no evidence that dbf4-NA109 caused premature Cdc14p release in a strain deleted for F081, which is thought help sequester Cdc14p in the nucleolus (Figure 20B). These data suggest that Dbf4p is not specifically inhibiting the FEAR pathway. The budding yeast SPOC prevents premature exit from mitosis in part by inducing Kin4p phosphorylation of Bfa1p, which antagonizes the Polo-dependent inhibition of the Bfa1p-Bub2p GAP [8, 9, 55]. Premature exit from mitosis in dbf4- NA109 dyn1 and dbf4-NA109 kar9 double mutants suggests that Dbf4p regulation of Polo is critical for robust cell cycle control in response to nuclear mispositioning. What remains unclear however, is whether this Dbf4p activity is regulated following activation of the SPOC. For instance, Cdc7p-Dbf4p may inhibit Polo to buffer against premature release of Cdc14p during late S-phase or early M-phase whether or not the SPOC is activated. Therefore, in the absence of the Dbf4p-Polo regulation Polo may prematurely activate the MEN before a nuclear orientation defect is sensed by the SPOC. Following APC-CchOp and APC-th1p activation during anaphase onset and exit, we suggest that degradation of Dbf4p provides a positive feedback loop for full Polo activation of the MEN and ultimately, cytokinesis. 134 Figure 20. (A) Deletion of the Dbf4p PIR does not enhance nucleolar segregation in cdc5-1 cells. DBF4, dbf4-NM 09, cdc5-1 and cdc5-1 dbf4-NA109 cells transformed with a centromeric EGFP-Nop1 plasmid were arrested in G1 with mating pheromone and released into the cell cycle in YPD at 34 degrees Celsius. Alpha-factor was added back after budding to permit a single cell cycle. Samples were taken at the indicated time points and scored for the presence of one or two GFP signals by florescence microscopy. (B) Deletion of the Dbf4p PIR does not enhance Cdc14 release when combined with deletion of F081. fob1A CDC14-EGFP (M3149) and fob1A dbf4-NA109 CDC14-EGFP (M3148) were arrested in G1 with mating pheromone and released into the cell cycle at 30 degrees Celsius. Alpha-factor was added back after budding to follow a single cell cycle. Samples were taken at the indicated time points and scored for release of Cdc14 from the nucleolus. 135 +%budded +°/o N0p1 segragation 100:wr 1oo.dbf4-NA109 l’ 80: 80: g 60: 60- 20- 20. o 04 " a so ab 160 150 a so 3'0 160 1&0 cdc5-1 - 100: 1oocdc5 1 dbf4-NA109 so- 80- m . . f, so: 60- § 40:! 4o- 20- 20‘ 0 , 0‘ 60 80 100120140 00‘ 60 80 100120 140 136 Figure 20 Cont. + % budded —I— % Cdc14 release Afob1 Afob1 dbf4-NA109 100 - 100 - ' m 801 80« E 60: 60: g 40: 4o: 20 - 20 - . r 0 . . '2" or. 30 60 90 120 on 30 60 90 120 137 How does Dbf4p inhibit Polo and mitotic exit? Dbf4p did not influence Polo protein levels or overall kinase activity. Dbf4p nonetheless inhibited Polo activity since dbf4 mutants unable to interact with Polo significantly suppressed the cdc5- 1 temperature sensitivity. Since the cdc5-1 allele retains significant Polo protein expression, Bfa1p phosphorylation and overall Polo kinase activity at the non- permissive temperature [37, 63], the primary cd05-1 MEN defect is in a Bfa1p- independent requirement for MEN pathway activation, perhaps Dbf2p activation or some other MEN-dependent step. Our observation that the dbf2-1 temperature sensitivity was also suppressed by loss of the Dbf4p PIR suggests that Dbf4p may specifically inhibit Polo activation of Dbf2p kinase independently of Bfa1p- Bub2p phosphorylation. Dbf2p was recently shown to promote cytoplasmic Cdc14p localization following MEN activation [69]. In addition, deletion of both BU82 and the DBF4 PIR suppressed the cdc5—2 ts better than either single mutant alone. In other words, since dbf4-M1109 further suppressed the ts phenotype of a cdc5-2 bub2A strain, this supports the contention that Dbf4p regulates MEN activity independently of Bfa1p-Bub2p. Deletion of the Dbf4p PIR also did not allow rebudding in the presence of spindle poisons as seen in bubZA mutants, again suggesting that Dbf4p plays a minor or redundant role to inhibit Polo phosphorylation of Bfa1p-Bub2p. Cdc7p-Dbf4p kinase phosphorylated the Polo PBD in vitro suggesting that Cdc7p-Dbf4p may antagonize Polo substrate binding. This possibility is consistent with the requirement for the PBD for targeting Polo to sites of MEN activity. Polo, Cdc15p, Dbf2p and Bfa1p-Bub2p are localized to SPB prior to 138 activation of the mitotic exit network [47]. Thus, we favor a model whereby Cdc7p-Dbf4p kinase inhibits precocious Polo binding to critical MEN substrate(s) by phosphorylating the Polo PBD. It will be interesting to investigate whether the Cdc7p-Dbf4p inhibition of Polo is regulated by cell cycle checkpoints and to determine the precise activity of Polo that is affected. MATERIALS AND METHODS: Images in this dissertation are presented in color. Construction of Yeast Strains, Plasmids and Baculoviruses Strains and plasmids used in this study are listed in Tables 1 and 2. PJ69—4a cells (MA Ta trp1-901 Ieu2-3,-112 ura3-52 his3-200 gaI4A gal80A LYSZ::GAL1- HIS3 GAL2-ADE2 met::GAL 7-IacZ) were used for two-hybrid experiments. All other strains were derivatives of W303 (MA Ta adeZ-1 trp1-1 can1-100Ieu2-3, - 112 his3-11, -15 ura3-1). Construction of Dbf4p N-terminal truncation mutants was previously described [26]. Cdc14-EGFP was constructed as described [70]. bub2A strains were created by replacement of the BUBZ ORF via homologous recombination with Sacl-Clal bub2A::URA3 fragment from pTR24 (A. Hoyt). The BAR1 ORF was deleted by homologous recombination with linearized pZV77 containing bar1A::LEU2 (B. Futcher). Construction of KAR9 and DYN1 deletions were previously described [8]. For yeast two-hybrid analyses, a Gal4 DNA binding domain (GBD) fusion to Dbf4p 67-227 was constructed by PCR amplification of Dbf4p residues 67-227 (Ncol-Pstl) and cloned into pGBKT7 (Clonetech). Deletion of 71bp within the 139 TABLE 1. Yeast strains used in this study Strain ' Genotype Source W303- 1A MA Ta ad92-1, ura3-1 his3-11, -15 trp1-1 Ieu2-3, -112 cam-100 R. Rothstein PJ69-4A MATa trp1-901 Ieu2-3, -112 ura3-52 his3-200 gal4A gal80A L YSZ: GAL 1-HIS3 GAL2-ADE2 met2:: GAL 7-lacZ E.A. Craig RHC15 W303 MA Ta mad2A::URA3 A. Murray M1261 W303 MA Ta dbf4-NA 1 09 C. Gabrielse M319 W303 MA Ta dbf2-1 This study M331 W303 MA Ta cdc15-2 M358 W303 MA Ta mcm2-1 M565 W303 MA Ta cdc14-1 M609 W303 MA Ta cdc14-3 M1585 W303 MA Ta CDC5-HA-URA3 M1614 W303 MA Ta cdc5-1 M1652 W303 MA Ta bub2A::URA3 M1656 W303 MA Ta dbf4-NA 1 09-kanMX 6 M1804 W303 MA Ta dbf4-NA 1 09 Cd05-1 M1860 W303 MA Ta bub2A.'.'URA3 dbf4-NA109- kanMX6 M1864 W303 MA Ta mad2A::URA3 dbf4-NA109- kanMX6 140 Table 1 Cont. M1866 W303 MA Ta dbf4-NA 1 09-kanMX 6 cdc 1 4-1 M1868 W303 MA Ta dbf4-NA109-kanMX6 cdc14-3 M1870 W303 MA Ta dbf4-NA109-kanMX6 dbf2-1 M1872 W303 MA Ta dbf4-NA1 09-kanMX 6 cdc15-2 M1918 W303 MA Ta kar9A::kl Trp1 M1992 W303 MA Ta CDC 1 4-E GFP-kanMX 6 M2005 W303 MA Ta CDC 14-EGFP-kanMX6 dbf4- NA 1 09-kan MX 6 fl M2007 W303 MA Ta dbf4-NA65-kanMX6 M2137 W303 MA Ta CDC14-EGFP-kanMX6 cdc5-1 M2139 W303 MA Ta CDC 14-EGFP-kanMX6 dbf4- NA 1 09-kanMX 6 cdc5- 1 M2179 W303 MA Ta kar9A::k/Trp1 dbf4-NA109-kanMX6 M2180 W303 MA Ta kar9A.‘.'kl Trp1 dbf4-NA109— kanMX6 M2269 W303 MA Ta dyn1A::lerp1 M2280 W303 MA Ta kin4A.'.'his5 dbf4-NA109—kanMX6 M2283 W303 MA Ta bub2A.'.‘HIS3 dbf4-NA109-kanMX6 Cdc5-2::URA3 M2285 W303 MA Ta bub2A.':HIS3 dbf4-NA109-kanMX6 cdc5-2::URA3 141 Table 1 Cont. M2287 W303 MA Ta cdc5-1 CDC14-EGFP-kanMX6 M2289 W303 MA Ta bub2A::H/S3 cdc5-2:.‘URA3 M2291 W303 MA Ta bub2A::Hl83 cdc5-2:.‘URA3 M2293 W303 MA Ta dyn1A::k/Trp 1 dbf4-NA 1 09- kanMX 6 M2295 W303 MA Ta dyn 1 A.'.'kITrp1 dbf4-NA 1 09- kanMX 6 M2357 W303 MA Ta CDC5-3HA-URA3 bar1A::LEU2 M2359 W303 MA Ta CDC5-3HA-URA3 dbf4-NA109- kanMX6 bar1A::LEU2 M2712 W303 MA Ta karQA::kITIp1 dbf4-NA109-kanMX6 cdc5-1 M2728 W303 MA Ta dyn1A::k/Trp1 bub2A.‘.‘H/S3 M2741 W303 MATa CDC5-15MYC 3HA-CDC7 M2743 W303 MA Ta CDC5-15MYC 3HA-CDC7 dbf4- NA109-kanMX6 M2748 W303 MA Ta CDC5-4XGFP-lerp1 SPC42- eqFP-hthT1 M2750 W303 MATa CDC5-4XGFP-kITrp1 SPC42- eqFP-hthT1 dbf4-NA109-kanMX6 M2804 W303 MA Ta dbf4-NA82-88-kanMX6 142 Table 1 Cont. M2818 W303 cdc5-1/cdc5-1 M2822 W303 cdc5-1/cdc5-1 DBF4/dbf4-NA109- kanMX 6 M2826 W303 cdc5-1/Cdc5-1 dbf4-NA 1 09-kanMX6/dbf4- NA 1 09-kanMX 6 M2908 W303 MA Ta cd05-1 dbf4-NA82-88-kanMX6 M3027 W303 MA Ta CdC55A.‘.’hi35 M3028 W303 MA Ta cdc55A.'.'his5 dbf4-NA109-kanMX6 M3093 W303 MATa [pMHY193; pRS316-GFP-Nop1] M3094 W303 MA Ta dbf4-NA109-kanMX6 [pMHY193; pR8316-GFP-Nop1] M3095 W303 MA Ta cdc5-1 dbf4-NA 1 09—kanMX 6 [pMHY193; pRS316-GFP—Nop1] M3096 W303 MATa cd05-1 [pMHY193; pRS316—GFP- Nop1] M3148 W303 MA Ta A fob 1 dbf4-NA 1 09-kanMX 6 CDC 1 4-E GFP-kanMX 6 M3149 W303 MA Ta Afob1 CDC 1 4-EGFP-kanMX 6 M3155 W303 MA Ta kin4A:.'hi85 cdc55A.'.'his5 dbf4- NA 1 09-kanMX 6 M3161 W303 MA Ta PDS1-HA3 DBF4-Myc18 143 Table 2. Plasmids used in this study. Plasmid Description Source pCG10 pRS415-DBF4 110-704 This study pCG52 pGBKT7-DBF4 67-227 “ pCG53 pGBKT7-DBF4 67-704 “ pCG60 pGBKT7-DBF4 67-227 A-732 to -802 ADH1 promoter “ pCG61 pCG60-DBF4 110-227 “ pCG68 pGAD-CDCS 422-705 “ pCG74 pGBKT7-DBF4 110-704 “ pCG166 pGAL-DBF4 1-225 “ pCG163 pGAL-DBF4 109-225 “ pCG167 pGAL-DBF4 65-225 “ pCG213 pGAL-DBF4 1-225 A 82-88 “ pCM1 pGAD-CDC5 422- 705 H641A, K643M “ pCM3 pGAD-CDC5 454- 705 “ pCM4 pGAD-CDC5 510-705 “ pCM16 pAcSG2-3myc-CDCS 65-705 “ pCM21 pCG60-DBF4 67-109 “ pCM24 pCM21 S84A, S92A, T95A, T105A “ pGAD- pGAD-CDC5 421-705 “ CDC5.3 pHS4 pSUMO-DBF4 66—109 “ pMW489 pRS41 5-DBF4 .. 144 Table 2 Cont. pMW535 pRS415-CDC5 “ pMW537 pGEX-KG-CDCS 357.705 “ pYJ38 pCGBO-DBF4 67-227 A82-88 “ pYJ1 50 pRS425-DBF4 1 10-705 “ pYJ152 pRS425-DBF4 482-88 “ pYJ154 pRS425-DBF4 .. pYJ161 pRS425-DBF4 40312-704 .. pYJ162 pRS425-DBF4 A65 + AC312-704 “ l - pYJ163 pRS425-DBF4 4109 + 40312-704 .. pYJ164 pRS425-DBF4 4182-88 + AC312- 704 “ pYJ214 pGBKT7-DBF4 A65 + 482-88 “ ADH1 promoter sequence of pGBKT7 (-647 to -717 from the ATG) removed a Rap1 p binding site and reduced the strength of GBD-Dbf4p 67-227 expression (which was otherwise lethal) to give pCG60. Point mutations and deletions were generated by site-directed mutagenesis using the QuikChange system (Stratagene). For all mutations, the entire coding sequence was verified by DNA sequencing. Construction of baculovirus plasmids encoding WT, NA6SDbf4p, NA1090bf4p, NA221Dbf4p and HA-Cdc7p was previously described [26]. The baculovirus transfer plasmid containing 3Myc—NA65Polo was constructed in pAcSGZ (BD Biosciences). High-titer baculoviruses were generated by 145 transfection of Sf9 cells using the BaculoGold kit (BD Biosciences) followed by plaque purification and virus amplification. For in vitro interaction assays, DNA encoding Polo amino acids 357-705 were PCR amplified with BamHl-Xmal linkers and cloned into pGEX-KG for expression of GST-Polo PBD in E. coli. The region encoding Dbf4p amino acids 66-109 was PCR amplified from pMW489 with Bsal-BamHl linkers and cloned into pSUMO (LifeSensors Inc.) for expression of Sumo-Dbf4p 67-109. Growth Media and Cell Cycle Experiments Cells were cultured in YPD (1% yeast extract, 2% bacto peptone, 2% glucose). Synchronous G1 cultures were obtained after addition of 5ug/ml (0.1ug/ml in bar1A cells) alpha-factor to cells for 3 hours. DNA content was analyzed by flow cytometry as previously described [17]. Drugs were added directly to plates immediately before pouring. Two-hybrid Experiments PJ69-4a cells containing pCG60 were transformed with a S. cerevisiae two- hybrid library. Interacting clones were recovered on medium lacking tryptophan, leucine, and histidine but containing 2 mM 3-aminotriazole at 30 degrees Celsius. Positive interactors were streak-purified and also tested for ADE2 reporter activity. Prey plasmids that activated both HIS3 and ADE2 expression were confirmed by retransformation in PJ69-4a and then sequenced. To quantify two- 146 hybrid interactions, co-transformed cells were spotted at ten-fold serial dilutions on selective media and grown for 2-3 days. Yeast Whole-cell Extract Preparation, Immunoprecipitation and Blotting Yeast protein extracts were prepared for Western blotting by trichloroacetic acid extraction [71] or for immunoprecipitation (IP) by bead-beating in NP-40 lysis buffer (20mM Tris-HCI, 150mM NaCl, 0.5% NP-40 and 1mM EGTA). HA-tagged proteins were immunoprecipitated using anti-HA monoclonal antibody (120A5) conjugated to protein A-Sepharose. Blots were probed in phosphate-buffered saline containing 0.1% Tween and 1% dried milk. 12CA5 (1 :1000) was used to detect HA-tagged proteins, 9E10 (1 :1000) to detect Myc-tagged proteins, and polyclonal sera against Cdc7p (1 :4000) and Dbf4p (1 :1000) were used to detect those proteins. Co-immunoprecipitation from Sf9 Insect Cells Sf9 cells were co-infected with HA-Cdc7p, 3Myc-NA65Polo and Dbf4p derivatives and then immunoprecipitated as previously described [26]. Whole cell extracts and lPs were probed with polyclonal antibodies against Cdc7p (1 :4000) and Dbf4p (1 :1000) as described above. 3Myc-NA65Polo was probed with 9E10 monoclonal antibody against Myc (1 :1000). Protein Expression, Purification and GST-Pull down 147 Cdc7p-Dbf4p kinase was purified as described [17]. GST or GST-PBD was induced in BL21 cells for 3 hours at 37 degrees Celsius using 0.5mM IPTG. Cells were sonicated in PBS containing 1% Triton X-100 and GST proteins were purified from soluble extracts by binding to glutathione-agarose (Amersham), elution in (20mM Tris-HCI, 150mM NaCl, 1mM EDTA and 10% glycerol) containing 5mM glutathione followed by dialysis against the same buffer. 6His- tagged Sumo and Sumo-Dbf4p were expressed in BL21 cells and extracted in HEPES extraction buffer (50mM HEPES-KOH, pH 7.5, 150mM NaCl, 2M Urea and 10% glycerol). Proteins were loaded onto a Ni++ column and washed (20mM HEPES-KOH, pH 7.5, 200mM NaCI and 10% glycerol) before elution using an J imidazole gradient. For GST pull-downs, Sumo, Sumo-Dbf4p, GST and GST- Polo were incubated with glutathione-agarose in the presence of buffer (20mM Tris-HCI pH 7.0, 300mM NaCl, 0.5% NP-40 and 1mM EGTA) for 1 hour at 4 degrees Celsius. The glutathione agarose beads were washed extensively and bound proteins separated on 12.5% SDS-PAGE gels. Blots were probed with polyclonal antisera raised against GST-Polo PBD (1 :1000) and yeast Smt3p (Sumo) (1 :1000). Kinase Assays For in vitro kinase assays, purified HACdc7p-Dbf4p (100ng) [17] was incubated with GST or GST-Polo PBD (300ng) at 30 degrees Celsius in kinase buffer (50mM Tris-HCI pH 7.5, 10mM MgCl2, 1mM DTT, 100uM ATP and 10uCi of [y- 148 32 P] ATP) for 20 minutes. Proteins were separated on 10% SDS-PAGE and visualized by autoradiography. Fluorescence Microscopy For direct fluorescence analysis of Cdc14-EGFP, cells were fixed in 3.7% formaldehyde at room temperature for 1 hour. DNA was stained using DAPI (1mg/ml) for 10 minutes at room temperature. For the experiments in Figure 14A, the absence of a distinct Cdc14-EGF P fluorescent signal (but accompanied by diffuse nuclear and cytoplasmic fluorescence) was scored as “released.” Any cell that had a distinct Cdc14-EGFP nucleolar fluorescence was counted as sequestered. Spindle morphology was detected after spheroplasting cells and incubation in methanol/acetone prior to incubation with antibodies: rat anti-tubulin (YOL1/34 Accurate Chemicals, 1:10) and goat anti-rat FITC (Jackson lmmunoresearch, 1:50). Cells were imaged using a 60x objective. Cdc5 Protein Abundance, Kinase Activity and SPB Localization To analyze Cdc5 protein abundance during the cell cycle K6019 and M1874 were arrested with alpha-factor (Spg/ml) for 3h, released into the cell cycle at 30°C in YPD and TCA extracts were analyzed by Western blotting. Cdc5-HA3p kinase activity was analyzed in asynchronous, G1 (0.1ug/ml alpha-factor), HU (0.2M) and nocodazole (15ug/ml) arrested cultures. Bead-heated whole cell extracts from strains M2357 and M2359 were made in (20mM Tris-HCI, pH7.4, 150mM NaCl, 0.5% NP-40, 1mM EGTA), immunoprecipitated with 12CA5-protein 149 A Sepharose and washed 4x in the same buffer. Kinase activity was measured on half of the IP using casein as a substrate in (50mM Tris-HCI, pH 7.5, 10mM McCl2, 5mM DTT, 2mM EGTA, 100mM ATP, 10uCi [y—32P] ATP). Bound proteins were separated on 10% SDS-PAGE and visualized by autoradiography or by Western blotting. To examine Cdc5p SPB localization, G1 arrested cells (M2748, M2750) were released into the cell cycle at 20 degrees Celsius in YPD media containing 15uglml nocodazole. Cells were fixed in 4% paraformaldeyde for 10 minutes, stained with DAPI (mg/ml) and analyzed for Cdc5-4xGFP spindle pole body localization using fluorescence microscopy. ‘ Nucleolar Segregation Assay To assess nucleolar segregation DBF4, dbf4-NA109, cdc5-1 and cd05-1 dbf4- NA109 cells transformed with a centromeric EGFP-Nop1 plasmid (pMHY193) were arrested in G1 with mating pheromone and released into the cell cycle in YPD at 34 degrees Celsius. Alpha-factor was added back after budding to permit a single cell cycle. Samples were taken at the indicated time points and scored for the presence of one or two GF P signals by florescence microscopy. Dbf4 Cell Cycle Abundance M3161 expressing Pds1-HA3p and Dbf4-Myc18p was arrested in G1 at 25 degrees Celsius with mating pheromone and released into the cell cycle at 20 degrees Celsius. Samples were taken at the indicated time points. Protein extracts were made for Western blotting by the TCA method [71] and cells were 150 processed for DNA content analysis by flow cytometry [17]. Western blots were probed using 9E10 (1 :1000) and 120A5 (1:1000) antibodies to detect Dbf4- Myc18p and Pds1-HA3p, respectively. ACKNOWLEDGMENTS: We thank A. Amon, E. Craig, L. Hartwell, A. Hoyt, L. Johnston, A. Murray, K. Nasmyth, E. Schiebel, A. Sugino and B. Stillman for yeast strains and plasmids; D. Morgan for communicating results prior to publication; C. Miranti and V. Schulz for technical assistance with microscopy; H. Scott for construction and purification of Dbf4p SUMO-derivatives; D. Dornbos for strain construction; C. Fox for comments on the manuscript; A. Amon, G. Pereira, F. Uhlmann and members of the Weinreich lab for helpful discussions. I wish to specifically acknowledge the following individuals for their scientific data and intellectual contributions that are directly contained within this manuscript. Their hard work and collegiality greatly assisted me in driving this research forward. Michael Weinreich: For his intellectual contributions and assistance with experimental design throughout all of this research and his primary role in writing and assembling the Miller et al. 2009, PLoS Genetics manuscript into publishable form and for the IP kinase assay included in Figure BB and contributions in Figure 8A. i would also like to thank him for strain and plasmid construction and the purification of Cdc7-Dbf4 kinase used in Figure 8D. Carrie Gabrielse: I am indebted to Carrie for making the initial discovery, by yeast two-hybrid screen, that the N-terminus of Dbf4 interacts with Cdc5 and that the 151 region preceding amino acid 109 is important for this interaction. Carrie contributed many of the constructs contained within Figure 6A and 6B and also generated most of the constructs and viruses in Figure 8A. She also contributed the cell cycle analysis in Figure 108 and data in Figure 11A and 118. I would like to acknowledge Ying-Chou Chen’s primary discovery that the Dbf4 amino acid 82-88 region is necessary for the interaction with Cdc5. This discovery was made independent of my research and we appended several of the figures within the Miller et al. manuscript to reflect this discovery. Ying-Chou Chen also contributed data in Figures 6A, 68, 10A and 11C. Chapter 1 was accepted and published by PLoS Genetics: PLoS Genet 5, e1000498. 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Mol Cell Biol 14, 923-933. 159 CHAPTER TWO Cch Phosphorylation Site Screen and Isolation of Phosphorylated Cdc5 lsoforms for Mass Spectrometry 160 ABSTRACT: The Dbf4-dependent kinase (DDK) is essential for replication initiation in eukaryotic cells. In addition to a role in S-phase, DDK inhibits mitotic exit by interaction and inhibition of the polo kinase Cdc5; however, the mechanism of this regulation remains unknown. Disruption of the Dbf4-Cdc5 interaction has no affect on Cdc5 catalytic activity, protein abundance or SPB localization. In vitro kinase analyses suggest that the Cdc5 PBD is a substrate of Cdc7-Dbf4. Here I identify Cdc5 PBD residues required for in vitro phosphorylation by DDK. Substitution of residues T532, 3573 and 8630 within the PBD with alanine impairs phosphorylation on the Cdc5 PBD. In addition, substitution of nearby residues within a conserved PBD fold had no effect on Dbf4-Cdc5 binding, suggesting that the loss of phosphorylation did not result from a loss of protein- protein interaction. In addition, DDK phosphorylated PBD isoforms were successfully resolved by PAGE to facilitate the physical mapping of DDK Cdc5 phosphorylation sites by mass spectrometry. INTRODUCTION: Activation of the Cdc14 dual-specificity phosphatase triggers mitotic exit in Saccharomyces cerevisiae following sister chromatid separation and nuclear division [1-3]. The precise ordering of cell cycle events is crucial to this process and activation of the mitotic exit network (MEN) plays a key regulatory role in regulating Cdc14 activity [4, 5]. MEN activation is triggered by increased Tem1 GTP activity in telophase [6, 7]. The Bfa1-Bub2 complex antagonizes MEN 161 signaling prior to full activation of the mitotic exit network by enhancing Tem1- GTPase activity at SPBs and is inhibited by Lte1 [7-10]. Inactivation of Bfa1-Bub2 at SPBs increases the cellular Tem1-GTP concentration, activating the SPB- bound kinases Cdc15 and Dbf2-Mob1 [11, 12]. Phosphorylation of Cdc14 and Net1 within the nucleolus releases Cdc14 and initiates mitotic exit [13-15]. The polo kinase in budding yeast, Cdc5, is an essential regulator of mitotic exit; inactivation of Cch arrests cells in telophase with partially divided nuclei and active Cdk [4, 16-18]. This defect is consistent with the role of Cdc5 in regulating Cdc14 by the Cdc14 early anaphase release (FEAR) and MEN [18- 20]. Cdc5 phosphorylation of Bfa1-Bub2, presumably at SPBs, triggers Bfa1- Bub2 subunit dissociation and Tem1-GTP activation of Cdc14 regulators Cdc15 and Dbf2 [21, 22]. Interestingly, Cdc5 activity outside the MEN is required for robust Dbf2 activity in cells, although the precise mechanism underlying this requirement is unknown [5]. Cdc5 activity in the FEAR network may account for the additional requirement of Cdc5 activity outside the MEN. Cdc14 release from the nucleolus reverses Cdc15 inhibitory phosphorylation by CDK prior to MEN activation. Therefore, regulation of Cdc14 activity by Cdc5 in the FEAR may link the segregation of sister chromatids and spindle elongation with the faithful execution of mitotic exit. A recent report suggests that Cdc5 regulation of Cdc14 occurs indirectly by inactivation of Swe1 at the GZ/M transition [23]. Swe1 is the budding yeast homolog of the human Wee1 kinase. Swe1 phosphorylation inhibits CDK activity prior to Mih1/Cdc25 dephosphorylation at the beginning of mitosis and prevents Cdc14 release by inhibiting Cdk activity on Net1 [23]. 162 Although especially important in regulating mitotic entry in human and S. pombe, Swe1 plays only a minor role in budding yeast. Limited understanding of the temporal and spatial mechanisms regulating Cdc5 activity complicates a complete interpretation of its function during mitosis. Ectopic Cdc5 activity causes untimely Cdc14 release in the presence of active checkpoints, suggesting that Cdc5 must be tightly regulated throughout the entire cell cycle [19, 24]. Cdc5 activity in G1 is primarily regulated by APC-dependent ubiquitination and degradation [25, 26]. Cdc5 protein levels rise in S-phase and catalytic activation by Cdc28 follows shortly thereafter [26]. Localization studies of Cdc5 have identified the bud neck and spindle pole bodies as sites of Cdc5 localization [27, 28]. Interestingly, Cdc5 appears to localize to SPB in S-phase, long before Bfa1 phosphorylation and MEN activation, implying a mechanism for inhibition of Cdc5 MEN activity until late mitosis [29]. Similarly, the mechanism of Cdc5 FEAR regulation prior to anaphase onset is currently unknown. An intact Cdc5 PBD is required for direct interaction with Cdc14 and proper SPB localization, suggesting that substrate priming and/or PBD inhibition may regulate Cdc5 activity at certain subcellular locations [30, 31]. Recent work in our lab demonstrated a novel role for Dbf4 in regulating Cdc5 activity during the cell cycle [29]. We reported a small PBD interacting region (PIR) within the Dbf4 N-terminus that when deleted suppressed the growth defects of MEN hypomorphs and rescued Cdc14 nucleolar release in cd05-1 cells. In addition, cells containing the Dbf4 PIR prematurely exit mitosis following spindle misorientation, resulting in ploidy abnormalities. As a result we 163 now appreciate a role for Dbf4 in regulating Cdc5 function during mitotic exit. Preliminary experiments eliminated altered SPB localization and decreased Cdc5 catalytic activity as mechanisms underlying Dbf4 regulation of Cdc5 [29]. Our discovery that Cdc7 is required for Dbf4-dependent inhibition of Cdc5 and identification of Cdc5 as a substrate for Cdc7-Dbf4 raises the possibility that DDK phosphorylation of the PBD is a mechanism for regulation of yeast polo kinase [29]. In an effort to identify the specific residues phosphorylated by DDK, l screened PBD mutants that were modified at putative Cdc7-Dbf4 phosphorylation sites by an in vitro phosphorylation assay. Purified Cdc7-Dbf4 was incubated with PBD phospho—ablation mutants matching previously published DDK consensus sites [29]. I identified T532, S573 and $630 as being required for efficient phosphorylation by DDK. The effect of the $630A, $573A and T532A mutations on the Cdc5-Dbf4 interaction was evaluated by two-hybrid analysis and found to have no effect on protein-protein interactions. In addition, in vitro phosphorylation of the Cdc5 PBD by purified Cdc7-Dbf4 identified multiple phosphorylated isoforms of Cdc5 for analysis by mass spectrometry. Identification of these phosphorylated residues should facilitate the development of reagents to examine the temporal and spatial regulation of Cdc5 by Cdc7- Dbf4. RESULTS: Mutation of Cdc5 PBD residues eliminates Cdc7-Dbf4 phosphorylation. 164 We previously reported that the Cdc5 PBD is a robust substrate of Cdc7-Dbf4 [29]. As a result, we hypothesize that DDK phosphorylation of Cdc5 inhibits its function in mitotic exit. Although the exact consensus sequence phosphorylated by DDK is not well defined, several recently published reports investigating Cdc7 phosphorylation of MCM helicase subunits identified residues important for in vitro and in vivo phosphorylation [32-36]. In these studies, Cdc7-Dbf4 demonstrated a strong preference for acidic residues at the pS +1 position, with a partial preference for hydrophobic residues immediately preceding the phosphorylation site [32-36]. Experiments in our lab support these findings [M. Weinreich unpublished data]. In an effort to identify PBD residues phosphorylated by Cdc7, l screened six putative Cdc7-Dbf4 phosphorylation sites matching published Cdc7-Dbf4 consensus sites within the Cdc5 PBD. Of all the Ser/T hr residues within the Cdc5 PBD, six (S-S383-D, A-S479-E, S-T532-E, L-8573-E, L-S630-D and l-S647-D) most closely matched the published consensus for Cdc7-Dbf4 phosphorylation. To investigate the role of these residues as sites of phosphorylation, Ser-to-Ala and Thr-to-Ala mutants were assessed for phosphorylation by DDK. Individual GST-PBD mutants were constructed via site-directed mutagenesis, expressed, purified and incubated in the presence of purified Cdc7-Dbf4 and radioactive ATP (Figure 21). Mutation of residues S383, S479 and S647 to alanine had no effect on substrate phosphorylation by kinase. In contrast, T532A, S630A and to a lesser extent $573A all significantly reduced Cdc7-Dbf4 phosphorylation. 165 >1 '5 8 O x.ssasgs M 1' In In 19 O 0) w I- t!) 0) 0) Dbf4 - ...:- m-a " '3 dB 7 i Q - _ ...... .. v. Autorad. PBD ' ' Coomassie Figure 21. Substitution of PBD residues inhibits DDK phosphorylation of the Cdc5 PBD. Recombinant GST-PBD mutants were analyzed for Cdc7-Dbf4 substrate specificity by in vitro kinase assay. Mutant substrates were incubated with purified Cdc7-Dbf4 at 30 degrees Celsius for 30min. Proteins were resolved by PAGE (Cdc7 and GST-PBD proteins co-migrate) and phosphate incorporation measured by autoradiography. 166 .mm 059“. E 550% w_ 553.88 EmSE EmEou co=m>=om Emmy .w:_m_oo $0506 on “a 986 095 .9 P mEmELOmemzoo .mEmSE 0mm mouoéfiEou cozm>=om 3.3 can EmEou 96:5 <20 3.5-on 355.92 FEB vegan—mam: 99> $.60 Eczema: mouoéno or: 8.53:5 Co: on «.88 mec 3223333 0mm .NN 959". £1- :0... 9h- :0... 9...- Nmm Pmn. 0n. <82 STI|E|U|-¢ <38 .0253 3% <88 mEIll|Ul-v o o o 531% mellllllfllus. 8T||I|UI§ e e e 58> 9518.3 5:855 IN .....8 38» Ed 167 Mutation of Cdc5 PBD residues does not affect the Dbf4-Cdc5 interaction. Identification of several residues that disrupt robust DDK phosphorylation on Cdc5 raised the possibility that some or all of these residues might regulate the interaction between Cdc5-Dbf4. The Cch PBD is a highly conserved interaction domain composed of two sub-domains termed polo boxes and a single polo-cap. We hypothesized that mutation of residues within the PBD might significantly disrupt PBD folding and sufficiently disrupt DDK interaction with Cdc5. Serine 630 and Threonine 532 are located within P82 and P31 respectively. To investigate whether substitution of these amino acids might influence the Dbf4- Cdc5 interaction, putative phosphorylation sites (S630, T532 and S537) and adjacent amino acids within the Gal4 AD-Cdc5 422-705 (PBD) were substituted to alanine and the interaction with the Dbf4 polo box interacting region (PIR) analyzed by two-hybrid analysis. Cells were co-transformed with wild type or mutant GAD-PBD and DBD-Dbf4 67-227 constructs. Co-transformants were cultured and spotted at 10-fold serial dilutions on SC —trp -|eu and SC -trp —leu - ’ his with 2mM 3AT and grown for 3 days at 30 degrees Celsius. Mutant protein expression was assessed by western blot analysis (Figures 22 & 24). Non- expressed proteins were omitted from two-hybrid analysis (Figures 23 & 25). Interestingly, T532A, S537A and $630A substitutions all interacted with the Dbf4 PIR, suggesting that substitution of these sites was not sufficient to disrupt the interaction with Cdc5 in the two-hybrid system. In addition, all proteins with substitutions near T532 and S630 also interacted with Dbf4, suggesting that 168 ( < 2 i 2 2 2 2 m. '3 to o r- m In 0 In '0 II, ID IO U U U U U U 0 o a a a a a a a 2 2 2 2 2 2 2 < < < o o o o o o 6 3 or-Gal4 AD L ...... ...... ‘_ gfi—sAo-cocs Ponceau S . W.,: :3 Figure 23. Substitution of PBD residues decreases GAD-Cdc5 expression. 'Western blot of various GAD-Cdc5 PBD mutants used in Figure 22. GAD-Cdc5 protein was probed using anti-Gal4 AD antibodies. Total protein expression was measured by staining with Ponceau S. Mutant Gal-PBD fusion proteins are indicated by arrow. 169 the Cdc5-Dbf4 interaction is not sensitive to sequence perturbations within these Cdc5 regions. Prior to these studies, we observed that S630E mutants failed to complement the deletion of CDC5 in cells; this is consistent with our model for Cdc5 regulation by Cdc7-Dbf4 and suggested that phosphorylation of this site might inhibit the essential function of Cdc5 during the cell cycle (unpublished data; M.W.). l analyzed the effect of phospho-mimetic substitutions of T532, S537 and S630 on the Dbf4-Cdc5 interaction using the two-hybrid system (Figure 22 & 24). Expression of $630E and T532E was not detectable, suggesting that substitution of these sites might destabilize domain folding (Figure 23 & 25). Together these results suggest that disruption of the Cdc5-Dbf4 protein interaction by substitution of putative phosphorylation sites is unlikely the cause of decreased DDK phosphorylation of Cdc5. Isolation of phosphorylated isoforms of Cdc5 for mass spectrometry. To isolate DDK-dependent phospho—specific isoforms of the Cdc5 PBD for use in mass spectrometry, recombinant full-length yeast HA—Cdc7 and Dbf4 were co- expressed in Sf9 insect cells and purified prior to in vitro kinase reactions with recombinant Cdc5 PBD protein. To maximize our chances for detecting a phosphorylation shifted isoform following kinase reactions, we wanted to use the lowest molecular weight form of recombinant PBD possible. I found that the original GST-PBD (~66 kDa) was highly resistant to proteolytic cleavage within 170 .mm 059“. 5 $505 2 568.56 EmSE EmEou co=m>=om v60 82200 $0606 om «m 986 025 L8 h 933 5622:. IN 250 .93 g m ... o 171 GAD-CDC5 SS3‘IA GAD-C005 T532A GAD-CDC5 GAD i GAD-C065 8573A ' GAD-CDC5 T532E ,1 l | GAD-CD05 £53311 ' :GAD-CDCS 35735 ’ b 1"“ or-Gal4 AD - . l I r i 1 Ponceau S I» Figure 25. Substitution of PBD residues decreases GAD-Cdc5 Expression. Western blot of various GAD-Cch PBD mutants used in Figure 24. GAD-Cdc5 protein was probed using anti-Gal4 AD antibodies. Total protein expression was measured by staining with Ponceau S. 172 the peptide linker, preventing me from using the PBD alone (~34kDa) as a substrate for DDK (data not shown). As an alternative, I utilized recombinant 6HlS-PBD (~45kDa) (Gift of Y.C. Chen). Following optimization of kinase reaction conditions (Magnesium and ATP concentration, incubation time, and kinase/substrate concentrations), active DDK was incubated with purified 6HlS- PBD in the presence of cold or radioactive ATP. Resolution of kinase reaction products by SDS-PAGE and silver staining identified a phosphorylation- dependent shift in Cdc5 mobility following incubation with active kinase (Figure 26). Three independent phosphorylated Cdc5 isoforms were visible following resolution by PAGE, suggesting that multiple DDK phosphorylation sites are present on Cdc5. A DDK consensus site following the HlS6 tag in our construct raises the possibility that this site is phosphorylated in our experiment, but the presence of multiple phosphorylated bands suggests that it is not the only residue being phosphorylated. Phosphorylated bands have been isolated and submitted for analysis by mass spectrometry. DISCUSSION: Consistent with the observation that the Cdc5 PBD is an effective substrate for in vitro DDK phosphorylation, we were able to identify specific residues within the PBD that when mutated decreased kinase phosphorylation. Of the six residues matching published Cdc7-Dbf4 consensus sites, we identified three sites (S630A, T532A and $573A) that resulted in a possible reduction in phosphorylation by DDK. Surprisingly, mutation of two of these sites individually (S630A or T532A) 173 Dbf4 + 1' Cdc7 1' 1' HIS-PBD ' 1' om C dc7 Autorad HIS-PBD HIS-PBD -..-5* Silver Figure 26. Resolution of phosphorylated Cdc5-PBD by SDS-PAGE. HIS-tagged Cch PBD was phosphorylated by purified Cdc7-Dbf4 and separated from unphosphorylated substrate by PAGE. Phosphorylated Cch was gel extracted and submitted for phosphopeptide mapping by mass spectrometry. (*) Shifted Cdc5 protein isoforms. 174 apparently caused complete loss of phosphorylation. This suggested that all three of these sites were not direct targets of DDK. To explain this finding, we proposed that one or more of these substitutions might disrupt the interaction with Dbf4, thereby indirectly affecting phosphorylation by DDK. Published reports indicate that the polo box domain is a phospho—dependent interaction domain that demonstrates strong preference for substrates with serine and proline at the pS -1 and +1 amino acid positions respectively [37-39]. Critical to this interaction are three highly conserved residues within polo box 1 (W517) and polo box 2 (H641 and K643) that contribute to phosphate binding. Histidine 641 and lysine 643 pincer residues coordinate direct binding with the negatively charged phosphate and tryptophan 517 is largely responsible for conferring specificity for serine at the pS -1 position [37, 39]. Interestingly, we have shown that the Dbf4-Cdc5 interaction occurs independent of phosphate binding and substitution of the conserved pincer residues (H641 and K643) has no affect on this interaction [29 and Y.C. and C. M. unpublished data]. Nonetheless, we hypothesized that disruption of these or flanking residues within conserved folding regions of the PBD might affect the interaction with Dbf4. We tested this by two-hybrid analysis using a Dbf4 N- terminal bait construct containing the polo box interaction region and Cch-PBD prey constructs containing mutations of interest. Surprisingly, the S630A, T532A and S573A mutant constructs all interacted with Dbf4, suggesting that a simple loss of Dbf4-Cdc5 interaction was not likely responsible for the observed loss of phosphorylation. Additionally surrounding residues D631A, T633A, $531A and 175 E533A also did not affect the Dbf4-Cdc5 interaction. Mutant residues preceding S630 (E628A and L629A) were not adequately expressed in the two-hybrid system, suggesting that these sites may contribute to protein stability. Although these results suggest that mutation of S630, T532 and 8573 does not influence the interaction between Dbf4-Cdc5, it is possible that these mutations may affect protein conformation differently as a GST-PBD construct. Analysis of the interaction between mutant GST-PBD proteins with Dbf4 may be informative in this regard, nevertheless, these studies suggest that significant progress in the identification of Cdc5 phosphorylation sites will more likely come through the physical mapping of sites by mass spectrometry. Phosphorylation of recombinant Cdc5 PBD protein for mass spectrometry. The results from our screen of putative DDK phosphorylation sites on Cdc5 strongly suggests that there are multiple DDK sites on the PBD. Initial attempts to map these sites on phosphorylated GST-PBD constructs were unsuccessful (CM. and MW. unpublished data). Identification of phosphorylated residues on proteins requires sufficient site occupancy of the target protein, which we may have now achieved by specifically isolating phosphorylated (shifted) Cdc5 In an effort to resolve phospho-specific PBD protein I utilized a smaller molecular weight 6HlS-PBD construct and phosphorylated recombinant protein using purified Cdc7-Dbf4. Using this technique, two shifted Cdc5 bands were easily discernible in 6HlS-Cdc5 preps incubated in the presence of kinase (Figure 26). Radioactive in vitro kinase assays confirmed that shifted bands, in 176 addition to the primary fast-migrating band, all incorporated radioactive phosphate (Figure 26). This suggests that DDK phosphorylates Cdc5 on multiple sites and implies that mutation of a single residue (as in Figure 21) would not be sufficient to completely eliminate DDK phosphorylation. Our current knowledge of the role Dbf4 plays in inhibiting Cdc5 during the cell cycle is complicated byour poor understanding of the specific timing and mechanism for Dbf4 inhibition of Cdc5. The absence of a biochemical marker to assess Dbf4 inhibition of Cdc5 represents a significant hurdle to our complete understanding of this interaction. This work provides important progress towards this goal. Identification of DDK phosphorylation sites on Cch will facilitate the development of reagents to assess spatial and temporal Dbf4 regulation of Cdc5 and assist us in understanding the full significance Cdc5 regulation by Dbf4 during the cell cycle. MATERIALS AND METHODS: Construction of Yeast Strains, Plasmids and Baculoviruses PJ69-4a cells (MATa trp1-901 Ieu2-3,112 ura3-52 his3-200 gaI4A gal80A LY82::GAL1-HIS3 GAL2-ADE2 met::GAL 7-IacZ) were used for two-hybrid experiments. For yeast two-hybrid analyses, mutations were generated by site- directed mutagenesis of pGAD-Cdc5 422-705 using the Quikchange system (Stratagene). Quikchange deletion of 70bp within the ADH1 promoter sequence of pCG60 (position -802 to -732) including the Rap1 binding site reduced the strength of Gal4 DBD-Dbf4 67-227 expression as previously described [29]. 177 Construction of baculovirus plasmids encoding Dbf4 and HA—Cdc7 was previously described [40]. High-titer baculoviruses were generated by transfection of Sf9 cells using the BaculoGold kit (BD Biosciences) followed by plaque purification and virus amplification. Two-hybrid Experiments PJ69-4a cells were co-transformed with bait and prey plasmids. Double transformants were recovered on medium lacking tryptophan (bait) and leucine (prey). Co-transformants were streak-purified and tested for growth on SC -trp - leu —his plates containing 2 mM 3-aminotriazole. Interactions were confirmed by retransformation of plasmids. To quantify two-hybrid interactions, co-transformed cells were spotted at 10-fold serial dilutions on selective media and grown for 3 days at 30 degrees Celsius. Protein Expression and Purification Methods used for expression and purification of the Cdc7-Dbf4p complex were previously described [41]. BL21 cells were used for expression and purification of recombinant proteins out of E. coli. GST and GST-tagged Cdc5 PBD aa422-705 were expressed for 3 hours at 37°C and released from cells by sonication in PBS containing 1% Triton X-100. Binding to a glutathione-conjugated matrix purified GST-conjugated proteins. Protein was washed (20mM Tris-HCL, 150mM NaCl, 1mM EDTA and 10% glycerol), eluted with 5mM free glutathione and dialyzed. 178 Kinase Assays For in vitro kinase assays, purified recombinant Dbf4-HACdc7 (50ng) was co- incubated with GST-Cdc5 PBD (500ng) in the presence of kinase buffer (50mM Tris-HCI, 10mM McClz, 5mM DTT and 2mM EGTA) with 1000M cold ATP and 1000i of [y-32P] ATP for 30 min at 30 degrees Celsius. Proteins were separated on 10% SDS-PAGE and visualized by silver staining or autoradiography. Mutant screen experiments were repeated following repurification of independent mutant proteins. Table 3. Plasmids used in this study. Plasmid Description Source pCG60 pGBKT7-DBF4 67-227 A-732 to -802 ADH1 [29] promoter pGAD- pGAD-CDC5 PBD 422—705 [29] CDC5.3 pMW537 pGEX-KG-CDCS 357-705 [29] pCM37 pGEX-KG-CDC5 357-705 S647A This study pCM50 pGEX-KG-CDC5 357-705 $630A “ pCM52 pGAD-CDC5 422-705 $647A “ pCM54 pGAD-CDC5 422-705 E628A “ pCM55 pGAD-CDC5 422—705 L629A “ 179 Table 3 Cont. pCM56 pGAD-CDC5 422—705 $630A 6‘ pCM57 pGAD-CDC5 422-705 D631A 66 pCM58 pGAD-CDC5 422-705 T633A 66 pCM59 pGAD-CDC5 422-705 $630E 66 pCM60 pGAD-CDC5 422-705 S647A 66 pCM61 pGAD-CDCS 422-705 S531A 66 pCM62 pGAD-CDC5 422—705 T532A ‘6 pCM63 pGAD-CDC5 422-705 T532E 6‘ pCM64 pGAD-CDC5 422-705 E533A 66 pCM65 pGAD-CDC5 422-705 SS37A 66 pCM66 pGAD-CDC5 422-705 S537E 66 pFJ240 pGEX-KG-CDC5 357-705 S573A 66 pFJ244 pGEX-KG-CDC5 357—705 $383A 66 pFJ245 pGEX-KG-CDC5 357-705 T532A 66 pFJ252 pGEX-KG-CDC5 357.705 S479A 66 ACKNOWLEDGMENTS: I would like to thank F. Chang and Y.C. Chen for plasmid construction; Y.C. Chen for purification of 6HlS-PBD protein; C. Gabrielse, Y.C. Chen and M. 180 Weinreich for critical reading of this thesis chapter; M. Weinreich for assistance in planning experiments and members of the Weinreich lab for helpful discussions. 181 REFERENCES: 1. 10. Shou, W., Seol, J.H., Shevchenko, A., Baskerville, C., Moazed, D.. Chen, Z.W., Jang, J., Shevchenko, A., Charbonneau, H., and Deshaies, R.J. (1999). Exit from mitosis is triggered by Tem1-dependent release of the protein phosphatase Cdc14 from nucleolar RENT complex. Cell 97, 233- 244. Visintin, R., Craig, K., Hwang, E.S., Prinz, S., Tyers, M., and Amon, A. (1998). The phosphatase Cdc14 triggers mitotic exit by reversal of Cdk- dependent phosphorylation. Mol Cell 2, 709-718. Tinker-Kulberg, R.L., and Morgan, DO. (1999). Pds1 and Esp1 control both anaphase and mitotic exit in normal cells and after DNA damage. Genes Dev 13, 1936-1949. 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Cdc7p-Dbf4p kinase binds to chromatin during S phase and is regulated by both the APC and the RAD53 checkpoint pathway. Embo J 18, 5334-5346. 185 CHAPTER THREE Summary and Conclusions 186 SUMMARY AND CONCLUSIONS The essential function of DDK in eukaryotic cells is in regulating DNA replication initiation in S-phase [1, 2]. Recent structure/function analyses of conserved Dbf4 regions implicate motif M and C in regulating Cdc7 binding and catalytic activity and suggest that the Dbf4 N-terminus‘ may regulate additional functions in S-phase [3-6]. To gain a better understanding of the role of the Dbf4 N-terminus in regulating DDK function, we carried out a two-hybrid screen to f identify unknown N-terminal interacting proteins and rediscovered an interaction with the yeast polo kinase Cdc5 (Figure 6) and identified a novel function for Dbf4 in regulating Cch-dependent cell cycle functions within mitosis [7]. Given the important role for Polo kinases in regulating mitosis and cancer progression, this discovery suggests a more sophisticated role for Dbf4 in eukaryotic biology and raises the possibility that increased understanding of Dbf4-Cdc5 regulation might have important implications for human health. Initial discovery and analysis of the Cdc5-Dbf4 interaction by Hardy and colleagues identified a minimal 147 amino acid region within the Dbf4 N-terminus that was required for interaction with full length Cdc5 [8]. The current study improves substantially on this finding in several ways. We identified that Dbf4 interacts with the Cdc5 PBD and narrowed the Dbf4 PIR to 43 amino acids (67- 109) adjacent the Dbf4 BRCT domain (Figure 6). Identification of this smaller sequence first raised the possibility that Dbf4 binds the Cdc5 PBD independent of protein phosphorylation. The polo box domain is a highly conserved protein- targeting domain within the C-terminal region of Cdc5 and demonstrates binding 187 specificity for S-pTlpS-P sites on substrates [9-11]. Mutation of potential Cch phosphopeptide binding sites within the Dbf4 PIR did not affect the Dbf4-Cdc5 interaction (Figure 6) and pulldown experiments using recombinant Cdc5 and Dbf4 proteins purified from E. coli recapitulated the Dbf4-Cd05 interaction (Figure 8). These observations support a phosphorylation-independent role for Cch- Dbf4 binding and have important implications for understanding polo kinase regulation. Presently, the Cdc5 residues required for Dbf4 binding have not been defined. Preliminary results suggest that Cdc5 binds Dbf4 using alternate residues than those used for phosphopeptide binding (Y.C. Chen, unpublished results). Future identification of the Dbf4 binding site on Cdc5 has the potential to provide additional insights into the regulation of polo kinases in cells. Cdc5 has many roles in mitosis, including its essential function in regulating Cdc14 during mitotic exit (Figure 3). This work identifies a significant function for Dbf4 in regulating this pathway. Importantly, this is the first report of a role for DDK in mitosis and suggests a regulatory function for Cdc7-Dbf4 in controlling cell cycle exit. Our observations that Dbf4 inhibits Cdc14 release and that deletion of the Dbf4 PIR bypasses the spindle position checkpoint provide evidence that DDK activity persists well after DNA replication (Figures 14 & 18). What remains unclear is whether Dbf4 inhibition of Cdc5 is regulated directly or indirectly by spindle position checkpoint activation. The SPOC checkpoint protein Kin4 inhibits Cdc5 MEN activation at spindle pole bodies in response to mispositioned spindles. It is unclear whether Dbf4-dependent inhibition of Cch is a target of this pathway or if Dbf4 indirectly affects mitotic exit during spindle 188 misorientation by global inhibition of Cdc5 function. Further experiments addressing this question will have important implications for our understanding of Dbf4-Cdc5 regulation during mitosis. Future studies have the potential to focus on several critical issues, which should continue to enhance our understanding of Dbf4 and Cdc5 cell cycle regulation. In particular, the specific mechanism by which Dbf4 inhibits Cdc5 function is of considerable interest. Genetic analyses of cdc5-1 suppression in dbf4 mutants that do not interact with Cdc7 suggest that both DDK subunits are required for inhibition of Cdc5 function (Figure 11C). This raises the possibility that DDK phosphorylation of Cdc5 may inhibit its function in mitotic exit. My observation that DDK phosphorylates Cch in vitro and determination of putative sites of phosphorylation suggest this may be the possible (Figure 8D & Figure 21). The identification of DDK phosphorylation sites using mass spectrometry will allow us to test the model that Cdc7-Dbf4 phosphorylates Cdc5 to inhibit PBD function and would provide a marker to assess the temporal and contextual regulation of Cdc5 by Dbf4. In addition, although my initial studies have focused on phosphorylation of the Cdc5 PBD, Cdc5 residues outside this region might also be targets of DDK phosphorylation. Cdc5 influences Cdc14 function at multiple mitotic stages. In this work, we provide strong evidence that Dbf4 regulates Cdc5-dependent release of Cdc14, independent of Bfa1-Bub2 MEN signaling. 1 identified that Cdc5 localization to SPBs (Figure 128), the site of Bfa1-Bub2, is unperturbed in dbf4-NA109 cells and provided genetic evidence for additional regulation of Cdc5 in bub2A 189 (hyperactive MEN) mutants (Figure 15). This raises the possibility that Dbf4- dependent regulation of Cdc14 may act through an alternative pathway/s. While this dissertation was in preparation, it was proposed that Cdc5- dependent regulation of the FEAR pathway occurs through inhibition of Swe1 (Wee1 homolog), resulting in enhanced CDK activity and promoting Cdc14 release through dissociation with its inhibitor Net1 [12]. CDK activity is essential to activate Cdc14 during mitotic exit as well, presumably by maintaining Net1 in an inhibitory state. This raises the possibility that Dbf4 may regulate Cdc5- dependent mitotic exit through inhibition of Net1. Interestingly, Cdc55-PP2A is thought to directly antagonize CDK in this function [13]. Deletion of CDC55 results in rebudding in the presence of spindle poisons, although to a much lesser extent than deletion of BUBZ (Figure 16 & [14]). Our observation that dbf4- NA109 failed to advance rebudding in cdc55A cells is consistent with a model whereby Dbf4 could inhibit Cdc5-dependent Net1 phosphorylation (Figure 16), however studies that Cdc5 also interacts with additional members of the FEAR network including Seperase and Slk19 implies that our current understanding of Cdc5 function in the FEAR remains incomplete [15]. Another untested hypothesis is the possibility that Dbf4 directly inhibits the Cdc5 interaction with Cdc14. A recent report suggests that Cdc14 and Cdc5 interact directly via the polo-box domain, possibly to modulate Cdc14 activity directly [15]. This model would be consistent with our previous data suggesting that Dbf4 regulates Cdc5 independent of Bfa1-Bub2. Although the significance of the Cdc14-Cdc5 interaction remains to be established, it raises the possibility 190 that inhibition of the Cdc5 PBD by DDK phosphorylation might prevent association of Cdc5 with Cdc14 and direct activation of the phosphatase in mitosis. Our lab’s initial discovery that deletion of the Dbf4 1-109aa region leads to complete loss of both the Cdc5 interaction and Dbf4 phosphorylation in response to HU treatment, raised the possibility that Rad53 might regulate Dbf4-dependent inhibition of Cdc5, possibly in response to DNA damage [5]. This hypothesis was complicated by the observation that dbf4-NA109 cells exhibit no growth phenotypes, compared to WT cells, in response to DNA damaging agents or spindle poisons [5]. These data combined with our cdc5-1 suppression experiments (Figure 10) suggest that Dbf4 regulates Cdc5 activity during every cell cycle, not just in response to checkpoint activation. Still, several published reports suggesting a direct interaction between Rad53 and Dbf4 and Rad53- dependent regulation of Cdc5 in response to DNA damage leave open the possibility that Rad53 and/or other checkpoint proteins may enhance Dbf4 regulation of Cch to coordinate events in S-phase with progression through mitosis [16, 17]. These studies will provide an important basis for future interrogation of the precise mechanisms of Cch regulation and provide a broader framework for our understanding of Cdc7-Dbf4 in the cell cycle. 191 REFERENCES 1. 10. Culotti, J., and Hartwell, L.H. (1971). Genetic control of the cell division cycle in yeast. 3. Seven genes controlling nuclear division. Exp Cell Res 67, 389-401. Johnston, L.H., and Thomas, AP. (1982). A further two mutants defective in initiation of the S phase in the yeast Saccharomyces cerevisiae. Mol Gen Genet 186, 445-448. Takeda, T., Ogino, K., Matsui, E., Cho, M.K., Kumagai, H., Miyake, T., Arai, K., and Masai, H. (1999). A fission yeast gene, him1(+)/dfp1(+), encoding a regulatory subunit for Hsk1 kinase, plays essential roles in S- phase initiation as well as in S-phase checkpoint control and recovery from DNA damage. Mol Cell Biol 19, 5535-5547. Ogino, K., Takeda, T., Matsui, E., liyama, H., Taniyama, C., Arai, K., and Masai, H. (2001). Bipartite binding of a kinase activator activates Cdc7- related kinase essential for S phase. J Biol Chem 276, 31376-31387. Gabrielse, C., Miller, C.T., McConnell, K.H., Deward, A., Fox, CA, and Weinreich, M. (2006). A Dbf4p BRCT-like domain required for the response to replication fork arrest in budding yeast. Genetics. Harkins, V., Gabrielse, C., Haste, L., and Weinreich, M. (2009). Budding Yeast Dbf4 Sequences Required for Cdc7 Kinase Activation and Identification of a Functional Relationship Between the Dbf4 and Rev1 BRCT Domains. Genetics. Miller, C.T., Gabrielse, C., Chen, Y.C., and Weinreich, M. (2009). Cdc7p- dbf4p regulates mitotic exit by inhibiting Polo kinase. PLoS Genet 5, e1000498. Hardy, C.F., and Pautz, A. (1996). A novel role for Cdc5p in DNA replication. Mol Cell Biol 16, 6775-6782. Elia, A.E., Rellos, P., Haire, L.F., Chao, J.W., lvins, F.J., Hoepker, K., Mohammad, D., Cantley, L.C., Smerdon, S.J., and Yaffe, MB. (2003). The molecular basis for phosphodependent substrate targeting and regulation of Plks by the Polo-box domain. Cell 115, 83—95. Elia, A.E., Cantley, LC, and Yaffe, MB. (2003). Proteomic screen finds pSer/pThr-binding domain localizing Plk1 to mitotic substrates. Science 299, 1228-1231. 192 11. 12. 13. 14. 15. 16. 17. Cheng, K.Y., Lowe, E.D., Sinclair, J., Nigg, EA, and Johnson, L.N. (2003). The crystal structure of the human polo-like kinase-1 polo box domain and its phospho—peptide complex. Embo J 22, 5757-5768. Liang, F., Jin, F., Liu, H., and Wang, Y. (2009). The molecular function of the yeast polo-like kinase Cch in Cdc14 release during early anaphase. Mol Biol Cell 20, 3671-3679. Queralt, E., Lehane, C., Novak, 8., and Uhlmann, F. (2006). Downregulation of PP2A(Cdc55) phosphatase by separase initiates mitotic exit in budding yeast. Cell 125, 719-732. Wang, Y., and N9, T.Y. (2006). Phosphatase 2A negatively regulates mitotic exit in Saccharomyces cerevisiae. Mol Biol Cell 17, 80-89. Rahal, R., and Amon, A. (2008). The Polo-like kinase Cdc5 interacts with FEAR network components and Cdc14. Cell Cycle 7, 3262-3272. Duncker, B.P., Shimada, K., Tsai-Pflugfelder, M., Pasero, P., and Gasser, SM. (2002). An N-terminal domain of Dbf4p mediates interaction with both origin recognition complex (ORC) and Rad53p and can deregulate late origin firing. Proc Natl Acad Sci U S A 99, 16087-16092. Sanchez, Y., Bachant, J., Wang, H., Hu, F ., Liu, D.. Tetzlaff, M., and Elledge, SJ. (1999). Control of the DNA damage checkpoint by chk1 and rad53 protein kinases through distinct mechanisms. Science 286, 1166- 1 171 . 193 APPENDIX An ectopic NLS sequence on dbf4-N4221 partially rescues cell growth. Coincident with experiments investigating the functional role of Dbf4 in regulating Cdc5 function, members of the Weinreich lab were completing a detailed structure-function analysis of the Dbf4 N-terminus [1]. Recent evidence suggested a role for the BRCT-like region (encoding motif N) in fission yeast Dfp1lDbf4 in maintaining cell viability during replication and DNA damage stress [2]. Building on this foundation, our lab identified additional sequences within Dbf4 proteins that differed significantly from other BRCT containing proteins and significantly affected Dbf4 function during replication stress [1]. To assist in bringing this project to completion, I conducted several experiments to help solidify our understanding of the role Dbf4 N-tenninal sequences contribute to normal Dbf4-Cdc7 function. Deletion of the Dbf4 N- terminal 221 amino acids caused significant delays in S-phase entry and replication [1]. To analyze the growth of this and other N-terminal Dbf4 mutants, strains were grown overnight to saturation and released into fresh YPD at low density (Figure A1). I recorded culture densities every hour following growth at 25 degrees Celsius. Deletion of N-terminal Dbf4 amino acids had a significant affect on cell growth compared to WT, dbf4-NA65 and dbf4-NA109 mutants. Previous experiments demonstrated S-phase initiation and progression defects in dbf4- NA221 mutants [1]. Addition of an ectopic NLS to dbf4-NLS-NAZ21 mutants partially overcame these defects and showed significant improvement in growth 194 suggesting that the Dbf4 BRCT domain did not affect Dbf4-Cdc7 replication activity. Initiation mutants show broad sensitivity to DNA damaging agents. Decreased origin activity and prolonged S-phase was a possible explanation for dbf4-NA221 sensitivity to a broad spectrum of DNA damaging agents. I compared the DNA damage phenotypes of several initiation mutants including cdc7-1, dbf4-1, cd06-1, cdc46-1 (mutant mcm5) and cdc47-1 (mutant mcm7) in addition to the dbf4-NA221 strain (Figure A2). Mutants were grown overnight and spotted at ten-fold serial dilutions on YPD with or without MMS, bleomycin or hydroxyurea and grown for 3 days at 25 degrees Celsius. All replication mutants were sensitive to MMS, suggesting that a decrease in origin efficiency may be responsible for this defect. The dbf4-NA221 mutant was sensitive to bleomycin and HU treatment and other mutants demonstrated varying phenotypes when grown on these compounds (Figure A2). Appending an ectopic NLS to the dbf4- NA221 mutant in an attempt to eliminate replication defects eliminated the sensitivity to MMS and bleomycin and suggested that the primary function of the BRCT region is response to replication stress (HU treatment) [1]. Dbf4 regulates Rad53 expression. Deletion of the Dbf4 N-terminal 221 amino acids increased HU sensitivity in cells raising the possibility that Dbf4 was inhibiting checkpoint function during replication stress. Experiments by other lab members, however, found that 195 cellular checkpoints, including Rad53, were not affected by this deletion [1]. Rad53 activation is accompanied by a characteristic gel mobility shift in the presence of HU. To determine whether this shift was present in Dbf4 mutants, asynchronous cultures of WT, dbf4-NA109 and dbf4-NA221 cells were incubated in the presence of HU for 0, 1.5 and 3 hours. Rad53-HA activation was examined by gel mobility shift (1.5 and 3 hours) after probing with anti-HA antibodies. As expected, incubation of HU still resulted in a Rad53 mobility shift in both mutants (Figure A3). More surprisingly, I observed decreased expression of Rad53 in the dbf4-NA221 mutant. Additional experiments by other lab members demonstrated that decreased Rad53 expression was independent of BRCT deletion and likely resulted from decreased overall Dbf4 activity. The significance of this finding remains uncertain, but raises the possibility that Dbf4 may counteract Rad53 activity during checkpoint activation. Acknowledgements I would like to acknowledge Michael Weinreich and Carrie Gabrielse for allowing me to contribute to the final stages of this project. All strains used in these experiments were constructed by MW. and CG. The above figures were included as part of the Gabrielse et al. manuscript and published in Genetics: Genetics. 2006 Jun;173(2):541-55. 196 1.0 ‘ 0.9 ' 0.8 - 0.7 1 0.6 1 0.5 - 0.4 ' OD. 600 0.2 ' Hours Figure A1. Growth curve of N-terminal Dbf4 mutants. WT (W303-1A), dbf4-N465 (M1642), dbf4-NA109 (M1261), dbf4-NAZ21 (M1356) and dbf4-NLS-NA221 (M1830) yeast strains were grown to saturation overnight then diluted in fresh YPD at equivalent optical densities. Strains were grown at 25 degrees Celsius and OD measurements recorded every hour. 197 Figure A2. Sensitivity of initiation mutants to DNA damaging agents. WT (W303- 1A), dbf4-N4221 (M1356), cdc7-1 (M199), dbf4-1 (M361), cdc6-1 (M378), cdc46- 1 (M323) and cdc47-1 (M317) yeast strains were spotted at ten-fold serial dilution on YPD with or without DNA damaging agents and incubated for 3 days at 25 degrees Celsius. ‘ 198 .. ... 9 O O Tnvobo . a o O C 792.60 .. p O O O 76660 .... O O O 73.6 m. o 0 O 768 . ,. t o O O FNN<2436 .....000 .fi. ..4. .3000; 925. {owned m2.)— Qovod c_o>Eoo_m DI Emd DI 5:.0 an; .253 199 U 0 O) ,8 WT NA109 NA221 C . 3 01.5 3 01.5 3 01.5 3 Hoursrn HU 1X M ' 5x -- ------ Rad53-HA 20X[ “‘1 ...... 4. ___..____% Non-specific Figure A3. Dbf4 regulates Rad53 expression. Asynchronous cultures of WT Rad53-HA (M1763), dbf4-NA109 Rad53-HA (M1792) and dbf4-NA221 Rad53-HA (M1763) indicated strains were grown in the YPD + 0.2M HU for 0-3 hours at 30 degrees Celsius. Protein extracts were probed with anti-HA antibody and exposed for increasing times (1X, 5X, 20X) to assess Rad53 expression. 200 REFERENCES 1. Gabrielse, C., Miller, C.T., McConnell, K.H., Deward, A., Fox, CA, and Weinreich, M. (2006). A Dbf4p BRCT-like domain required for the response to replication fork arrest in budding yeast. Genetics. 2_ Ogino, K., Takeda, T., Matsui, E., liyama, H., Taniyama, C., Arai, K., and Masai, H. (2001). Bipartite binding of a kinase activator activates Cdc7- related kinase essential for S phase. J Biol Chem 276, 31376-31387. 201 "11111111111111[[[ljljlljljlflljllll'es