. .u z .5 . 3-9.. V. c. I (v. I. :55 . .3 ah“? , a.» .9. ii. .- . 12.52..- it) I ,mth :1... u: B‘v’«v§ zo. run“... {9. A, rmmmnk» azfietufi .E \ ll A . ll... .1- , . 73.5.5.5: {Iszi 1:1 , . ‘ . , .5331. 9th! r . 3.. .h.‘ ;..‘....»....u.. , A 3:...» a iv? .t.nl.u...u- . Tia 3.2:, NA: .t.....fl .. \a . . .x. .ff ‘ [l “if r IIHIIIIIIIHHIII\llllllHIUIlHlHllllllllllllIIIIIIHHIIHI LIBRARY 31293 01770 064 Michigan State University ' This is to certify that the dissertation entitled MOLECULAR CLONING, BACULOVIRUS EXPRESSION AND RECONSTITUTION OF DROSOPHILA MITOCHONDRIAL DNA POLYMERASE presented by Yuxun Wang has been accepted towards fulfillment of the requirements for PhD. degree in Genetics Major professor Date 1/ 15/99 MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 PLACE IN REFURN BOX to remove this checkout from your record. TO AVOID FINE return on or before date due. MAY BE RECALLED with earlier due date if requeSted. DATE DUE DATE DUE DATE DUE MEG a 9% 2002 04”” 3 Mom 011:1’70‘5‘ Wllflfis 1M COMM“ MOLECULAR CLONING, BACULOVIRUS EXPRESSION AND RECONSTIUTION OF DROSOPHILA MITOCHONDRIAL DNA POLYMERASE By Yuxun Wang A DISSERTATION submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Genetics Program 1998 ABSTRACT MOLECULAR CLONING, BACULOVIRUS EXPRESSION AND RECONSTITUTION OF DROSOPHIIA MITOCHONDRIAL DNA POLYMERASE By Yuxun Wang Molecular cloning of the accessory subunit of Drosophila mitochondrial DNA polymerase (pol 1) was accomplished, and its mammalian homologs were identified. Sequence similarity and structural features that are shared between the accessory subunit of Drosophila pol y and its mammalian homologs suggest that the heterodimeric structure of pol y is conserved from insect to man. Co-expression of the catalytic and accessory subunits encoded by recombinant baculoviruses reconstitutes the heterodimeric pol 'y holoenzyme in both the mitochondria and cytoplasm of Sf-9 insect cells. Purified recombinant pol yexhiblts nearly identical physical and biochemical properties as the native enzyme from DrosOphila embryos. The roles of both subunits of Drosophila pol y in template-primer DNA recognition and binding were studied by photochemical cross-linking and DNase I footprinting. The catalytic subunit contains the DNA binding activity and the DNA-binding domain was mapped to a 65 kDa fragment. Native pol 7 was found to protect two helical turns (20 nt) of the primer strand when it forms a discrete and stable complex with template-primer DNA. Mitochondrial single-stranded DNA-binding protein was found to enhance the specific interaction between pol ‘y and template-primer DNA. To my wife, Yueying Cao and my son, Ruotao Wang iv ACKNOWLEDGEMENTS I would like to start by thanking my thesis advisor and mentor, Dr. Laurie S. Kaguni, for her guidance, encouragement, and support during the past five years of my graduate studies. I have learned not only science from her but also parenting and other very important things. I would also like to thank members of my committee: Drs. Zachary Burton, Tom Friedman, Shelagh Ferguson-Miller, Suzanne Thiem, and Vilma Yuzbasiyan- Gurkan for their continuing support and scientific input. I also want to thank Dr. Jon Kaguni for many helpful suggestions in our regular research group meeting, and Dr. John Wang for allowing me to use the cell culture equipment in his laboratory. Many thanks to members of both Kaguni laboratories: Dr. Anuradha Alahari, Carol Farr, Li Fan, Inmaculada Ruiz de Mena, Kevin Carr, Matias Vicente, and Lyle Simmons for correcting my English, sharing experimental materials and techniques, and interesting discussions. I am also very grateful to many former students of the Kaguni laboratories, including Dave Lewis, Mark Sutton, Carla Margulies, J ianjun Wang, Andrea Williams, Anthony Lagina, and Phillip Kiefer. I thank many fellow graduate students in the department for everything, especially Lei Lei for sharing success and frustration in research, and other interests. Finally, I thank my wife, Yueying Cao and my son, Ruotao Wang for their love, support, encouragement, and understanding. TABLE OF CONTENTS LIST OF TABLES ..................................................................... LIST OF FIGURES ..................................................................... LIST OF ABBREVIATIONS ..................................................................... CHAPTERI INTRODUCTION ....................................................................... Mitochondria ..................................................................... Mitochondrial DNA ........................................................ Mitochondrial DNA Replication .......................................... Mitochondrial DNA Mutations and Their Consequences ....................................................... Mitochondrial DNA Repair ............................................... DNA Polymerases .............................................................. Catalytic Mechanism ...................................................... Fidelity of DNA Strand Synthesis ...................................... Prokaryotic DNA Polymerases .......................................... DNA polymerase I ................................................ DNA polymerase II ............................................... DNA polymerase III .............................................. T7 DNA polymerase .............................................. T4 DNA polymerase .............................................. Eukaryotic DNA Polymerases .................................... DNA polymerase 0t ............................................... DNA polymerase B ............................................... DNA polymerase 5 ................................................ DNA polymerase e ................................................ DNA polymerase C ................................................ DNA polymerase y ................................................ DNA Polymerase Accessory Proteins ................................... Overview .......................................................................... vi Page ix xii CHAPTER II INTERACTION BETWEEN DROSOPHILA POL 7 AND TEMPLATE-PRIMER DNA. ............................................... 27 Introduction. ...................................................................... 28 Experimental Procedures ..................................................... 31 Materials ..................................................................... 31 Methods ..................................................................... 32 R e s u l t s ............................................................................... 35 Drosophila pol 7 forms a discrete complex with template-primer DNA ................................................. 35 The catalytic subunit of Drosophila pol 7 makes close contact with template-primer DNA ................................. 38 The recombinant catalytic subunit of Drosophila pol y is active in DNA binding .................................................. 41 A proteolytic fragment of the catalytic subunit retains DNA binding activity .............................................. 41 Cross-linked Drosophila pol y retains DNA polymerase activity ......................................................... 46 Drosophila pol y footprints two helical turns of its primer ........................................................... 49 Mitochondrial single-stranded DNA binding protein enhances template-primer DNA-binding of pol ‘y ....................... 49 Discussion .......................................................................... 52 Future Research ................................................................. 55 CHAPTER III MOLECULAR CLONING OF THE ACCESSORY SUBUNIT OF DR OS OPHILA POL y ............................................ 57 Introduction.. ...................................................................... 58 Experimental Procedures. ................................................... 60 Materials ..................................................................... 60 Methods ..................................................................... 61 Results ............................................................................... 64 Cloning of the B subunit of Drosophila pol y ........................... 64 vii Identification of human and mouse homologs of the B subunit of Drosophila pol 'y ..................................... 68 Genomic DNA structure of theDrosophila B subunit gene ............................................................... 74 Discussion.. ........................................................................ 79 Future Research ................................................................. 85 CHAPTER IV PRODUCTION AND BIOCHEMICAL CHARACTERIZATION OF RECOMBINANT DROSOPHILA POL y ............................... 87 Introduction.... ................................................................... 88 Experimental Procedures....... .............................................. 91 Materials ..................................................................... 91 Methods ..................................................................... 92 Results.... ........................................................................... 101 Construction of recombinant baculoviruses and verexpression of recombinant a and B subunits of Drosophila pol yin cultured insect cells .............................. 101 Mitochondrial import of recombinant 0t and B subunits ............................................................ 104 Co-expression of recombinant 0t and B subunits reconstitutes pol 'y holoenzyme ............................................ 107 Purification of recombinant pol y holoenzyme .......................... 110 Stimulation of both DNA polymerase and 3’-5’ exonuclease activities of recombinant Drosophila pol y by salt and by mtSSB ................................................ 116 Discussion .......................................................................... 1 17 Future Research ................................................................. 120 LIST OF PUBLICATIONS--YUXUN WANG .......................................... 121 REFERENCES ............................................................................... 122 viii LIST OF TABLES Page Chapter IV Table 1. Purification of recombinant Drosophila pol y from baculovirus-infected Sf-9 cells ................................................. 113 ix LIST OF FIGURES Chapter I Chapter II Figure 1. Binding and photochemical cross-linking of Drosophila pol y to template-primer DNA ..................................... Figure 2. Photochemical cross-linking of Drosophila pol y to template-primer DNA covalently links the catalytic subunit to primer DNA .................. Figure 3. Binding and photochemical cross-linking of the recombinant catalytic subunit of Drosophila pol y to template-primer DNA .............. Figure 4. Photochemical cross-linking of trypsin-digested Drosophila pol y to template-primer DNA ................................................... Figure 5. Cross-linked Drosophila pol y retains DNA polymerase activity ........... Figure 6. Drosophila pol y footprints two helical turns of primer DNA, and mitochondrial single-stranded DNA-binding protein enhances primer DNA binding by pol y ........................................ Chapter III Figure 1. Nucleotide and deduced amino acid sequences of the B subunit of Drosophila DNA polymerase y ................................... Figure 2. Sequence alignment of deduced amino acid sequences of the B subunit of Drosophila, man, mouse, and rat .......................... Figure 3. Putative structural features of the B subunit of Drosophila pol y and its human, mouse, and rat homologs ................................ Figure 4. Genomic DNA structure of Dr030phila pol “y .................................. Page 37 4O 43 45 48 51 66 7O 73 76 Chapter IV Figure l. Baculovirus transfer vectors .................................................... 94 Figure 2. Overexpression of recombinant 0t and B subunits in Sf-9 cells ....................................................................... 103 Figure 3. Mitochondrial import of recombinant a and B subunits ........................................................................... 106 Figure 4. Reconstitution of recombinant Drosophila pol y heterodimer in mitochondria of Sf—9 cells ..................................... 109 Figure 5. Co-expression of or and B subunits lacking mitochondrial presequences ..................................................... 112 Figure 6. Purification of recombinant Drosophila pol y ................................ 115 ATP ATPase BrU CDK dATP dCTP dGTP D. melanogaster DNA DNase I dNT P dRP d’I'I‘P E. coli mRNA mtDNA mtSSB NCBI Ni-NTA nt PCN A LIST OF ABBREVIATIONS apurinic/apyrimidinic adenosine triphosphate adenosine triphosphatase 5-bromouracil cyclin-dependent kinase deoxyadenosine triphosphate deoxycytosine triphosphate deoxyguanosine triphosphate Drosophila melanogaster deoxyribonucleic acid deoxyribonuclease I deoxynucleoside triphosphate deoxyribose phosphate deoxythymidine triphosphate Escherichia coli gene product kilodalton messenger RNA mitochondrial DNA mitochondrial SSB National Center for Biotechnology Information nickle nitrilo-tri-acetic acid nucleotide proliferating nuclear cell antigen xii pfu PMSF RF—A RNA RP-A S. cerevisiae S. pombe SSB ssDNA SV40 tRNA UV X. laevis plaque forming unit(s) phenylmethylsulfonyl fluoride replication factor C ribonucleic acid replication protein A Saccharomyces cerevisiae Schizosaccharomyces pombe single-stranded DNA-binding protein single-stranded DNA simian virus 40 transfer RNA ultra-violet Xenopus laevis xiii CHAPTER I INTRODUCTION MITOCHONDRIA Mitochondria are eukaryotic cellular organelles that contain a double membrane, their own DNA genomes, and an independent protein synthesis system. Their main biological function is energy production through oxidative phosphorylation and ATP production (Wallace, 1992). They also contribute to the biosynthesis of pyrimidines, amino acids, phospholipids, nucleotides, folate coenzymes, heme, urea and many other metabolites (W hittakr, 1978). Recently, mitochondria have been demonstrated to play an important role in the regulation of apoptosis (Mignotte and Vayssiere, 1998). The observation that modern mitochondria possess many characteristics of eubacteria has led to the hypothesis that they have evolved from eubacteria-like endosymbionts in a primitive eukaryotic cell as the host (Gray, 1989a). It is still not clear whether a eukaryotic or another prokaryotic cell served as the host for the ancestor of mitochondria. An alternative hypothesis is that an anaerobic and autotrophic archaebacterium served as the host for a eubacterium (the symbiont) and it was the common ancestor of all present day eukaryotic cells (Martin and Muller, 1998). Present day unicellular eukaryotes that do not contain mitochondria arose later by losing their mitochondria during evolution. Since taking residence in the host cell, mitochondria have diversified greatly and lost their genetic independence, and now requiring nuclear encoded proteins for oxidative phosphorylation, mitochondrial DNA replication, transcription, translation, and other biological functions (Schatz, 1996). However, mitochondria still contain their own genome, the mitochondrial DNA (mtDNA), which encodes a limited number of proteins. Several genetic and degenerative human diseases have been linked to specific mutations in mtDNA (Wallace, 19953), or mutations in nuclear genes encoding mitochondrial proteins (Casari, et al., 1998). Accumulation of mtDNA mutations in somatic cells has been hypothesized to be one of the causes of cellular senescence and aging because mitochondria have been shown to possess relatively limited DNA repair functions and mtDNA is physically in closer contact to DNA damaging reagents (Shadel and Clayton, 1997). Mitochondrial DNA More than 70 complete mitochondrial genomes that represent many diverse species including mammals, birds, and insects have been sequenced and are available from the public domain database (GenBank, NCBI). Mitochondrial genomes have evolved extraordinarily and they show remarkable differences in their sizes and structures (Gray, 1989b; Gray, et al., 1998). This likely derives from a variety of different evolutionary constraints. However, mtDNA from animals as diverse as Drosophila and mammals share many common structural features, such as (1) a circular double-stranded DNA of 16 to 19 kb, (2) compact genome organization, (3) polycistronic transcriptional units, and (4) an identical gene content (Leblanc, et al., 1997). Available data from plant mtDNA sequences show that they contain more respiratory protein genes, a set of ribosomal protein genes, are larger in size, and appear to be more dynamic in structure (Fauron, et al., 1995). Data accumulated during the past 15 years have revealed that linear mitochondrial DNAs exist in a large number of lower eukaryotes, mostly in ciliata and fungi (Nosek, et al., 1998). Because of the diversity of mtDNAs in terms of their size and structure and consequently different replicative mechanisms, this presentation will focus on studies of animal mtDNA replication, including insects (Drosophila) and mammals (man and mouse). Animal mitochondrial genomes are much smaller (approximately 16 to 19 kb) as compared to nuclear genomes that are usually close to or exceed a billion base pairs in size. However, this second non-nuclear genome is equally important for the viability of animals. Animal mtDNA usually contains one noncoding region which shows the most diversity in sequence and length, and contains the origin of replication and the transcriptional promoters (Larsson and Clayton, 1995). The protein coding capacity of animal mtDNA is well conserved from insects to mammals. The oxidative phosphorylation system requires more than 90 protein components, but only 13 polypeptides are encoded by the mitochondrial genome. These 13 mitochondrial genes encode the same set of protein subunits of the oxidative phosphorylation complex in animals (Leblanc, et al., 1997). In addition, animal mtDNAs all contain genes for two ribosomal RNAs and 22 tRNAs which are required for the translation of mtDNA-encoded mRN A (Wallace, 1995b). The tRNA genes are usually located between protein-coding genes and distributed almost randomly over the entire mtDNA, with some degree of clustering (Shadel and Clayton, 1997). Mitochondrial DNA Replication Duplication of any DNA genome is the result of the collaboration of many enzymes and other proteins. The basic mechanisms of this complicated event are well conserved from prokaryotes to eukaryotes, despite the evolutionary distance. From E. coli, phage T4, to mammals (both nuclear and mitochondrial), DNA polymerases are assisted by other enzymes and replication proteins to complete DNA replication with high speed, fidelity and processivity in a regulated fashion (Kornberg and Baker, 1992). In a typical animal cell that usually contains many mitochondria, mtDNA represents less than 1% of the total DNA, even though mtDNA molecules greatly outnumber the nuclear chromosomal DNA molecules. The total number of mtDNA molecules in a particular cell may vary according to the respiratory requirement, or the developmental stage, or the growth rate of the cell (Robin and Wong, 1988). The mtDNA copy number of mtDNA per mitochondrion is relatively constant. Thus, the variability in the total number of mtDNA molecules in different tissues is primarily the result of mitochondrial density (Veltri, et al., 1990). This suggests that mtDNA replication is a regulated event during mitochondrial biogenesis. However, replication of mtDNA is not coupled to cell cycle control in contrast with strictly regulated chromosomal DNA replication (Clayton, 1992). Data regarding the cis-acting DNA elements and trans-acting protein factors required for the initiation of animal mtDNA replication are mostly from vertebrates (Lee and Clayton, 1997; Lee and Clayton, 1998), especially mammals, although more evidence in other species is accumulating. Mitochondrial DNA replication in mammals, and likely in other animals, is asymmetric (Shadel and Clayton, 1997). Both leading and lagging DNA strands are synthesized continuously, and the origins for the two strands are mapped to different genomic locations. In mammals, the origin of leading strand synthesis is located within the noncoding region while the lagging DNA strand initiation site is located approximately two thirds the genomic distance away from the leading strand origin. The initiation of lagging strand replication does not occur until the replication fork originated from the leading strand origin has passed the lagging strand initiation site so that the lagging strand origin is in the single-stranded state. In Drosophila, lagging strand initiation occurs when leading strand replication is almost complete (Goddard and Wolstenholme, 1980). The current model for mammalian mtDNA replication is based upon biochemical analysis of early replication intermediates and mitochondrial RNA processing (Lee and Clayton, 1998). Mitochondrial DNA Mutations and Their Consequences Because each mitochondrion contains multiple copies of mtDNA and each cell contains many mitochondria, a single mutation does not cause immediate effect in cellular function. The deficiency of mitochondrial function will be observed first in those tissues requiring high energy production when the mutant form of mtDNA accumulates to a critical portion of the total mtDNA (Wallace, 1995b). However, animal mtDNA is so compact that almost every mutational event will have a high probability of occurring in either the coding region or essential regulatory sequences of mitochondrial genes. Eukaryotic cells have evolved mechanisms to maintain the genetic stability of mitochondrial genomes. Mutations involving large deletions and base substitutions have been identified in the mtDNA of humans and have been shown to associate with several maternally inherited and ageing-related neuro-muscular degenerative diseases ( MITOMAP data base; http://www.gen.emory.edu/mitomap.html). Large deletions in the non-coding region of mtDNA which contains the replication origin and promoters for transcription would be lethal in both germline and somatic cells. That is presumably the reason why these mutants have never been detected. However, more than 90 large deletions at different sites have been reported (Kogelnik, et al., 1998), and they fall into two classes. The first type of deletions always involve direct repeat sequences at the deletion junctions and deletions in the second class do not contain an obvious repeated sequence (Mita, et al., 1990). Mitochondrial DNA with large deletions apparently can not be transmitted through the female germline (Larsson, et al., 1992) because virtually all cases reported are sporadic. It is likely that a mitochondrion containing mutant mtDNA with large deletion is lethal during embryogenesis or female germline cell development. Deleted mtDNA was also observed to accumulate with age in many mammalian somatic cells (Cortopassi, et al., 1992; Cortopassi and Arnheim, 1990; Tanhauser and Laipis, 1995). Pathogenic point mutations have been reported at 28 sites in 9 protein-coding genes and 34 sites in 15 tRNA genes. They are associated with at least 16 clinical syndromes. Only two rRNA gene point mutations leading to disease have been observed (Kogelnik, et al., 1998). The presence of mtDNA pseudogenes in the nuclear genome makes the detection and interpretation of mitochondrial point mutation more difficult (Tsuzuki, et al., 1983). For example, whether point mutations in cytochrome c oxidase subunit genes (C01 and C02) can be linked to late-onset Alzheimer disease requires further studies (Davis, et al., 1997; Wallace, et al., 1997; Hirano, et al., 1997; Hutchin, et al., 1997). The fact that same mtDNA mutation in a protein-coding gene can produce distinct clinical syndromes in different families or individuals suggests that other factors likely contribute to the pathogenesis as well. These factors may include (1) the tissue-specific distribution of mutant mtDNA, (2) the severity of the deficiency in individual oxidative phosphorylation complexes, and (3) protein-protein interactions between mitochondrial and nuclear encoded subunits within a specific complex. Any structural change or lesion in DNA requires fixation to yield a mutation, and mutations can occur before or during DNA replication. Mitochondrial DNA polymerase (pol y) is the key enzyme in the replication of mtDNA and is possibly also involved in mtDNA repair (Longley, et al., 1998a). It certainly plays an important role in maintaining a low mutation rate in mtDNA. Mitochondrial DNA Repair Although DNA repair in animal mtDNA is not as well documented as that in the nuclear genome, accumulating evidence indicates clearly that DNA repair does occur in animal mitochondria (Driggers, et al., 1993; Ikeda and Ozaki, 1997; LeDoux, et al., 1992; LeDoux, et al., 1993). The apparently higher mutational rate (or evolution rate) in mtDNA is not necessarily the result of the lack of DNA repair functions. However, deficiency in DNA repair functions will certainly increase the overall mutation rate. Many different factors are likely to contribute to the overall higher mutation rate in mtDNA, such as multiple rounds of replication of mtDNA per cell cycle, a higher rate of fixation of mutation due to rapid genetic drift, close contact with DNA damaging agents such as oxygen free radicals, a relative higher frequency of direct repeat sequences in mtDNA, and the existence of single-stranded DNA over a relatively long period of time during mtDNA replication. Recently, a number of laboratories have reported the identification and purification of DNA repair and recombination activities/enzymes from mitochondria of several evolutionally divergent organisms (Chi and Kolodner, 1994; Takao, et al., 1998). The human recombinant catalytic subunit of pol 7 has been shown to contain a DNA repair activity (Longley, et al., 1998a). Thus, earlier speculation that mammalian mitochondria may lack DNA repair function (Clayton, 1982) was probably the result of insufficient experimentation. DNA POLYMERASES Many enzymes, including but not limited to DNA polymerase, primase, telomerase, DNA ligase, topoisomerase, helicase, nuclease, and other replication accessory proteins such as single-stranded DNA-binding protein, initiation factors, processivity factors, and termination factors are indispensable for the cell to replicate its genome completely (Kornberg and Baker, 1992). However, the major enzyme responsible for cellular DNA replication is DNA-dependent DNA polymerase (DNA pol). DNA polymerases also provide the pivotal function of DNA synthesis in the cellular processes of DNA repair and recombination. A large number of DNA polymerases have been studied and they exhibit a rich variety in their structures and functions (Kornberg and Baker, 1992; Wang, 1991). For example, the mammalian repair DNA polymerase B (pol B) is a single subunit enzyme of 39 kDa, and the replicative polymerase of E. coli, pol III, has 18 subunits, a molecular mass of close to 900 kDa, and multiple enzymatic activities (McHenry, 1988). However as a superfamily, they are unified by exhibiting an essential catalytic function: template/primer-dependent nucleotidyl transferase (Kornberg and Baker, 1992). The fundamental function of DNA pols is to extend the 3’ end of a primer strand by adding a single deoxynucleotide, and the difference in their structure is determined by requirements for other enzymatic activities such as primase and proofreading 3'-5' exonuclease, for high processivity in replication, for interacting with other replication proteins, and for regulatory control, e.g. cell cycle control. Prokaryotic organisms encode at least one DNA polymerase, and as many as seven DNA polymerases have been discovered in higher eukaryotes (Wang, 1991). Many viruses encode their own DNA polymerase to bypass the host replicative machinery. DNA pols are grouped into four families (family A, B, C, X) based upon their amino acid sequence similarity (Ito and Braithwaite, 1991). Typically, replicative DNA pols work in complex with other accessory proteins, which provide additional functions that help meet a number of requirements and overcome a variety of constraints inherent in the semiconservative duplication of long supercoiled and condensed double-helical DNA genomes. Catalytic Mechanisms Despite the diversity of DNA polymerases in their protein sequences, all of them contain a small number of conserved crucial amino acid residues that form the active site. DNA polymerases can not synthesize DNA de novo; they all require a template and a primer, synthesize DNA from 5' to 3' using deoxynucleoside triphosphate substrates. Although detailed structural data are available only from a small number of DNA polymerases (Kiefer. et al., 1997; Beese, et al., 1993; Wang, et al., 1997a; Wang, et al., 1996; Kim, et al., 1995; Eom, et al., 1996; Sawaya, et al., 1994; Sawaya, et al., 1997), the conservation of chemistry of DNA synthesis is well established. The reaction pathway of DNA polymerization has been characterized for several prokaryotic enzymes (Patel, et al., 1991; Kuchta, et al., 1987; Dahlberg and Benkovic, 1991; Capson, et al., 1992). Kinetic data were derived mainly from bacteria and phage enzymes because of the availability of large quantities of purified enzymes in these systems. However based upon large number of mutagenesis studies, the basic chemical mechanisms can be expected to be conserved in eukaryotic enzymes (Copeland and Wang, 1993; Jung, et al., 1990). The enzyme binds template-primer DNA first to form a binary complex that is able to productively bind a dNTP to form a catalytically competent ternary complex. A conformational change in the ternary complex directs both substrate DNA and nucleotide to the position for phosphodiester bond formation. A second conformational change following the chemical step releases the pyrophosphate and repositions the binary complex for the next incoming deoxynucleotide. Crystal structures of binary or ternary complexes containing DNA polymerase, template-primer DNA, and deoxynucleotide (Doublie, et al., 1998; Li, et al., 1998) have identified both the specific acidic residues that bind divalent metal and form the catalytic active site, and other residues participating in DNA and nucleotide binding. Fidelity of DNA Strand Synthesis Two separate processes ensure the high fidelity of the DNA synthesis reaction. High selectivity for the correct deoxynucleoside triphosphate by the DNA polymerase and proofreading activity by the 3’-5’ exonuclease combine to limit the error rate to 10'6 (Kunkel and Mosbaugh, 1989; Kunkel, 1992). The high selectivity comes from (1) discrimination against mispairs in dNTP binding, (2) higher resistance in the conformational change step when an incorrect nucleotide is bound, and (3) reduced efficiency in the chemical step of phosphodiester bond formation with a mispaired 3’-end . Again most data are from prokaryotic enzymes and the relative importance of each step varies between the polymerases studied (Dahlberg and Benkovic, 1991; Capson, et al., 1992). The contribution of kinetic selectivity in eukaryotic DNA polymerases remains to be 10 studied. Most, if not all, replicative DNA polymerases contain a proofreading 3’-5’ exonuclease activity either in a separate subunit or in the catalytic subunit. If an incorrect nucleotide is added to the growing 3’-end of primer strand, then the template-primer DNA becomes a better substrate for the 3’-5’ exonuclease instead of the 5’-3’ DNA polymerase. In studies with E. coli pol I, the second conformational change following the incorporation of an incorrect nucleotide is much slower, thus providing an opportunity for the enzyme to move the 3’-end containing the mismatch to the exonuclease site (Kuchta, et al., 1987). If the wrong nucleotide survives each of the error-reducing steps and the enzyme enters the next cycle of polymerization, the binding of the next dNTP is further reduced when the template-primer DNA has a mismatch at its 3’-end. This gives the enzyme another opportunity to transfer the mispaired DNA substrate to the proofreading active site. Alternatively, the enzyme dissociates from the DNA containing mispaired primer end, and the DNA becomes a substrate for another exonuclease. In addition to the DNA synthetic cycle per se, post-replication mismatch DNA repair and maturation of Okazaki fragments in lagging strand synthesis also contribute to the overall fidelity of DNA replication (Barnes, et al., 1996). Prokaryotic DNA Polymerases DNA polymerase I DNA polymerase I (pol I) from E. coli is a single—chain monomer with multiple functional domains which exhibit 5’-3’ DNA polymerase, and 3’-5' and 5’-3' exonuclease activities (Kornberg and Baker, 1992). Pol I is the most abundant DNA polymerase in bacterial cells, and was the first DNA polymerase discovered. It is the most extensively studied DNA polymerase and serves roles in DNA replication, recombination, and repair. 11 It is the only DNA polymerase that has 5'-3' exonuclease activity and is able to utilize nicked circular duplex DNA as a template, unwinding the parental DNA strand from its template as it polymerizes. Several crystal structures of the Klenow fragment of E. coli pol I bound to substrate DNA (Beese, et al., 1993) and structures of pol Is from other bacteria are now available (Kim, et al., 1995), providing insights into the enzymatic mechanism of both DNA polymerase and exonuclease activities. E. coli pol I is the prototype of family A DNA polymerases. DNA polymerase 11 DNA polymerase II (pol II) from E. coli was discovered many years ago, yet its precise role in pathways of DNA replication and repair remains undetermined. It has been established that dinA (polB), the structural gene encoding pol II, is repressed by LexA as a part of the SOS regulon, and that the cellular levels of pol II are increased by a factor of seven following induction of the SOS regulon (Bonner, et al., 1988). In spite of data suggesting that pol II could be involved in repair of UV-induced damage (Masker, et al., 1973), null mutants of p013 are viable and do not appear to exhibit an increased sensitivity to UV light (Escarceller, et al., 1994). Pol II can synthesize DNA with a high processivity in the presence of the E. coli pol HI accessory proteins, the B sliding clamp and the clamp loading complex in vitro. Recent genetic data suggest that pol 11 could be involved in DNA synthesis on the chromosome and on the F episome in actively dividing cells. Rangarajan et al. replaced the wild-type polB gene on the chromosome with a proofreading-defective polB mutant (polBexI) and found significant increases in mutation frequencies of reporter genes on both the chromosome and on F(lac) episomes (Rangarajan, et al., 1997). DNA pol II is a family B polymerase sharing sequence 12 similarity with eukaryotic pol 0t catalytic subunit and bacteriophage T4 DNA polymerase. The entire genome from the archaeon M. jannaschii has recently been sequenced (Bult, et al., 1996) and found to contain a single DNA polymerase gene which exhibits high degree of amino acid identity with E. coli pol H. Thus it appears that this E. coli pol II homolog must catalyze both replicative and repair DNA synthesis in M. jannaschii. DNA polymerase III Pol III holoenzyme from E. coli is responsible for the replication of the bacterial chromosome. Pol III holoenzyme is distinguished from other DNA polymerases in the cell by its high processivity (>50 kbp) and rapid rate of synthesis (750 nucleotides/s) (Kornberg and Baker, 1992; Kelman and O'Donnell, 1995). Pol HI holoenzyme is a large complex composed of 10 functional subunits encoded by 9 different genes, some of which are present in multiple copies for a total of 18 polypeptide chains (Onrust, et al., 1995; McHenry, 1988). The holoenzyme can be resolved into three primary functional units. (1) The DNA polymerase core contains three tightly bound subunits as follows: the polymerase or at subunit, the 3'-5' exonuclease or 8 subunit, and the 6 Subunit, with no known function. (2) The ring-shaped sliding clamp is a processivity factor consisting of a dimer of the B subunit. (3) The five protein 7 complex is the “clamp loader” that couples ATP hydrolysis to assembly of B clamps around the DNA substrate (Onrust, et al., 1995). The 7 complex consists of five different subunits (72515’ lxlwl). The 5 subunit is the major touch point to the B clamp and leads to ring opening, but is buried within the 7 complex such that contact with B is prevented (Naktinis, et al., 1995). The 7 subunit binds ATP but 13 is not an ATPase by itself (Tsuchihashi and Kornberg, 1989). The 8’ subunit bridges the 8 and 7 subunits, resulting in a 788’ complex that exhibits DNA-dependent ATPase activity and is competent to assemble clamps on DNA (Onrust, et al., 1991). Upon binding of ATP, a change in the conformation of the 7 complex exposes 8 for interaction with B subunit (Naktinis, et al., 1995). The x and \u subunits form a 1:1 701! complex and the x subunit makes direct contact to single-stranded binding protein to facilitate replication of an SSB-coated template (Kelman, et al., 1998; Glover and McHenry, 1998). A dimer of the I subunit acts as a “macromolecular organizer,” holding together two molecules of core and one molecule of 'ycomplex to form the pol III* subassembly (Onrust, et al., 1995). T7 DNA polymerase T7 DNA polymerase (T7 pol) is a two subunit enzyme. The catalytic subunit is the 80 kDa product of gene 5 of the phage, and it interacts physically with one host protein, E. coli thioredoxin to form a complex. The consequence of this interaction is to convert gene 5 protein from a polymerase with low processivity to one of high processivity (Tabor, et al., 1987). A 76-amino acid sequence (residues 258-334), unique to T7 pol, has been implicated in this interaction . In fact, insertion of this thioredoxin binding sequence into the homologous region of E. coli pol I has converted it into a polymerase of high processivity (Bedford, 1997). T7 pol does not require other accessory proteins to bind to the template-primer DNA substrate. T7 pol also interacts with the hexameric gene 4 protein of the phage, a protein that provides both helicase and primase activity at the replication fork to coordinate both leading and lagging strand synthesis (Park, et al., 1998). Both T7 14 pol and gene 4 protein in turn interact with the T7 gene 2.5 protein, a single-stranded DNA-binding protein that stimulates both polymerase and primase activities (Kim and Richardson, 1993). T7 pol also contains a N-terminal 3’-5’ exonuclease domain, and is a member of family A DNA polymerase (Ito and Braithwaite, 1991). The three-dimensional structure of T7 DNA polymerase has been solved recently (Doublie, et al., 1998), and it provides additional support for a universal DNA polymerization mechanism. T4 DNA polymerase Bacteriophage T4 gene 43 encodes the viral T4 DNA polymerase (T4 pol) with a molecular weight of 103 kDa. T4 pol has regions of high similarity with animal virus DNA polymerases and human DNA polymerase alpha catalytic subunit, and shares very limited similarity with E. coli pol I and no detectable similarity with T7 pol (Spicer, et al., 1988). It is therefore a member of the family B DNA polymerase group (Ito and Braithwaite, 1991). It resembles T7 pol in that it also contains both DNA polymerase and 3'- 5' exonuclease activity in its C- and N-terminal regions, respectively. The enzyme is also a sequence-specific RNA-binding autogenous translational repressor (Young, et al., 1992). Unlike T7 pol, T4 pol requires other phage-encoded replication accessory proteins to recognize effectively the template-primer DNA substrate. The product of gene 45 acts as sliding clamp and the products of genes 44 and 62 form a complex with DNA-dependent ATPase activity that functions as a clamp loader protein (Kroutil, et al., 1998). This is very similar to E. coli pol 111, although in E. coli pol III the functionally analogous proteins are subunits of the pol III holoenzyme. Eukaryotic DNA Polymerases Many years of biochemical and genetic studies have led to the identification of at 15 least 6 distinct DNA polymerases in eukaryotic cells, DNA pols or, B, y, 8, 8, and C (Wang, 1991). The S. cerevisiae genome contains an open reading frame that could potentially encode another DNA polymerase (Sugino, 1995). Recently a human gene that potentially encodes a seventh DNA polymerase has been identified (GenBank accession number AF052573, Sharief,F.S., Ropp,P.A., and Copeland,W.C.) and it is shown to be a homolog of the Drosophila Mus308 protein. The Drosophila mus308 gene has been implicated in DNA cross-link repair and has sequence similarity to both DNA polymerase and DNA helicase (Harris, et al., 1996). Whether these two genes actually encode a new DNA polymerase remains to be confirmed by biochemical analysis of their gene products. The only DNA polymerase located in mitochondria of animal cells is DNA pol 7, although a B-like DNA polymerase has been reported in the protozoan Crithidia (T orri and Englund, 1995). In addition, plant cells contain a y-like DNA polymerase for the replication of chloroplast DNA (Sala, et al., 1980). DNA polymerase 0t The highly conserved DNA polymerase tat-primase complex (pol a) is the only eukaryotic DNA polymerase that can initiate DNA synthesis de novo (Lehman and Kaguni, 1989). It is required both for the initiation of DNA replication at chromosomal origins and for the discontinuous synthesis of Okazaki fragments on the lagging strand at the replication fork (Wang, et al., 1989). Pol as isolated from yeast to mammals contain 4 subunits of 165-180, 70-86, 60, 50 kDa (Kaguni, et al., 1983b; Plevani, et al., 1988; Miyazawa, et al., 1993). Pol 0t provides RNA-DNA primers for the initiation of both 16 leading and lagging strand synthesis based upon the evidence from reconstituted SV40 DNA replication. The large catalytic subunit contains the DNA polymerase activity (Copeland and Wang, 1991), and the two small subunits form the primase. The p48 subunit contains the primase activity (Schneider, et al., 1998) while the p58 subunit is necessary for the stability and enzymatic activity of p48 subunit. The second-largest subunit of the mouse DNA polymerase at has been shown to facilitate both production and nuclear translocation of the catalytic subunit of pol 0t (Mizuno, et al., 1998). Genes for the catalytic subunit as well as those for other subunits have been cloned and mapped from many species including Drasaphila, human, mouse, and yeast. Post-translational modification of pol at has been observed in human and S. cerevisiae cells. Both the human p180 and p70 subunits are phosphorylated in a cell cycle- dependent manner and the replication activity of pol a is modified by phosphorylation (Nasheuer, et al., 1991; Voitenleitner, et al., 1999). These data suggest that the mitotic phosphorylation of pol or may affect its physical interaction with other replicative proteins and/or with DNA at the replication fork. Because pol or is the only DNA polymerase that can synthesize DNA de nova, the phosphorylation of pol or may play critical regulatory role in the initiation of DNA replication. DNA polymerase B Pol B from man, mouse, rat, and cow, is a single-subunit enzyme of 39 kDa and belongs to the family X DNA polymerase group (Ito and Braithwaite, 1991). The yeast pol II gene encodes a protein with predicted molecular mass of 68 kDa which is a homolog of 17 mammalian pol B. Mammalian pol B consists of 2 domains linked by a protease-sensitive hinge region. This enzyme is an integral part of the base excision repair pathway (Sobol, et al., 1996). The C-terminal 31 kDa domain possesses the polymerase catalytic activity. The N-terminal 8-kDa domain is required for single-stranded template DNA binding and has been recently shown to also contain an activity to excise deoxyribose phosphate (dRP) groups from 5'-incised apurinic/apyrimidinic (AP) sites during base excision repair (Prasad, et al., 1998; Matsumoto, et al., 1998). P0] B is so far the only eukaryotic DNA polymerase for which a crystal structure is available (Sawaya, et al., 1997). The polymerase active site of pol B is structurally similar to that of prokaryotic DNA polymerases, suggesting a universal DNA polymerization mechanism(Sawaya, et al., 1994). Expression of the pol B gene is constitutive under normal growth conditions because pol B enzymatic activity and mRNA levels are independent of cell growth and cell cycle regulation. There have been reports that pol B gene expression can be induced by DNA damaging agents, consistent with its role in DNA repair. DNA polymerase 8 Pol 8 has been shown to play important roles in DNA replication, nucleotide excision repair, base excision repair and V-D-J recombination of immunoglobin genes by both genetic and biochemical approaches. The catalytic subunit of pol 8 is well conserved from yeast to human, although the precise subunit structure of pol 8 is still an unresolved 18 issue. P01 8 is usually purified as a heterodimeric enzyme of a 125 kDa catalytic subunit and a 50 kDa small subunit from mammalian cells, but budding yeast pol 8 has been shown to contain a third subunit (Burgers and Gerik, 1998). P01 8 purified from the fission yeast S. pombe contains five distinct subunits, three of which are encoded by essential genes (Zuo, et al., 1997). The p125 subunit contains both DNA polymerase and 3'-5' exonuclease activity. An active human recombinant pol 8 heterodimer has been reconstituted in the baculovirus expression system. The small subunit of human pol 8 is required for the interaction and stimulation of DNA polymerase activity by proliferating cell nuclear antigen (PCNA) (Zhou, et al., 1997). Genes encoding both subunits have been cloned from many species. DNA polymerase 8 Like pol 8, the exact subunit composition of pol e is not yet resolved. Purified human pole contains at least two subunits of 255 kDa and 55 kDa. In S. cerevisiae, pol 8 is reported to consist of a 256 kDa catalytic subunit and four additional subunits of 80, 34, 31, and 29 kDa. The 34 and 31 kDa subunits are encoded by the same gene and may result from post-translational processing. The exact role of pol e in DNA replication is not clear (Bambara, et al., 1997). In yeast, the gene encoding the catalytic subunit is essential for cell growth (Sugino, et al., 1998), but pol e is not required for in vitro replication of SV40 virus DNA (Zlotkin, etal., 1996). DNA polymerase C 19 The genes encoding pol C, rev3 and rev7, have been cloned based on their phenotypes in a UV sensitivity assay in budding yeast S. cerevisiae (Morrison, et al., 1989) . Pol C, has been shown to be a nonessential DNA pol in yeast, and is involved in bypass DNA synthesis on DNA templates containing lesions (Nelson, et al., 1996). The rev3 gene product is the catalytic subunit of pol C and rev7 protein constitutes an accessory subunit which stimulate the overall DNA polymerase activity. Mammalian homologs of the catalytic subunit have been identified (Gibbs, et al., 1998), and it is likely that the pathway and mechanisms of DNA-damage induced mutagenic DNA synthesis are conserved. DNA polymerase “y Pol y is a low abundance enzyme in somatic cells, usually representing less than 1% of total cellular DNA polymerase activity. Nevertheless , it is an essential enzyme for the cell viability. P01 7 purified from many species shares many biochemical features including, the size of the catalytic subunits, intrinsic proofreading 3'-5' exonuclease activity, the capacity to use homoribopolymers as substrate in vitra, sensitivity to N— ethylmaleimide and dideoxynucleotides, and resistance to aphidicolin (Wemette and Kaguni, 1986). Molecular cloning of catalytic subunit genes has shown that the catalytic subunit of pol 'y from yeast to man is well conserved in amino acid sequence, and that it belongs to the family A DNA polymerase group (Lecrenier, et al., 1997). However, the sequence similarity between pol y and other family A DNA polymerases is restricted to the catalytic domains of both polymerase and exonuclease, and 'y pols share unique conserved sequences among them, thus the ypols represent a distinct subfamily. 20 Drosaphila pol ‘y in its native form purified from embryos is a heterodimer of 125 kDa (0t) and 35 kDa (B) subunits (Wemette and Kaguni, 1986). It has been shown to synthesize DNA efficiently on a variety of DNA substrates, and is stimulated by high salt concentration and by mitochondrial single-stranded DNA-binding protein (mtSSB). The large 125 kDa catalytic subunit contains both 5’-3’ DNA polymerase and 3’-5’ exonuclease activities (Lewis, et al., 1996) while no specific biochemical function has been assigned to the 35 kDa subunit. It has been shown that the B subunit is important to maintain the catalytic efficiency and/or the structural integrity of the holoenzyme (Olson, et al., 1995). In one report pol 'y purified from human Hela cells contained polypeptides of 140 and 54 kDa (Gray and Wong, 1992). In another, the catalytic subunit (140 kDa) was shown to copurify with a complex of four polypeptides (77, 52, 49, and 45 kDa) (Longley, et al., 1998b). The purified porcine liver pol 7 contains four polypeptides of 120, 55, 50, and 48 kDa (Kunkel and Mosbaugh, 1989), and a preparation from Xenapus laevis embryos possesses polypeptides of 100, 85, 55, 40, and 31 kDa in association with the 140 kDa catalytic subunit (Insdorf and Bogenhagen, 1989a). DNA Polymerase Accessory Proteins DNA polymerase processivity factors have quite unique names in different systems because of their initial characterization. In E. coli, the B subunit of pol III holoenzyme serves as the processivity factor. For bacteriophage T4, gp45 protein provides the same biological function. In eukaryotes, PCNA is conserved from yeast to man and it increases the processivity of pol 8 even though it was originally discovered as a cell cycle-regulated 21 nuclear protein. The crystal structures of PCNA and the B subunit have been solved (Kong, et al., 1992; Krishna, et al., 1994). These three proteins do not share significant sequence homology. However, their closed circular structures are very similar. While the B subunit of E. coli pol III is a dimer, PCNA and T4 gp45 protein form homotrimers. All three proteins contain a hole in the center that encircles the duplex DNA thus allowing them to track along the DNA molecule. These sliding clamps do not contain DNA-binding activity but depend upon clamp-loading proteins (replication factor C in eukaryotic systems, 7 complex of pol III in E. coli, and gp42/66 complex in phage T4) to be loaded on DNA. Once they are loaded on DNA, these sliding clamp proteins are able to physically interact with replicative DNA polymerases thus conferring high processivity to the holoenzymes at the replication fork. PCNA has also been shown to bind many other proteins, including FEN-l nuclease , DNA ligase I, cyclin-dependent kinase(CDK) inhibitor protein p21, nucleotide excision repair protein XPG, DNA-(cytosine 5) methyltransferase, mismatch repair proteins MLH1 and MSH2, and cyclin D (Kelman and Hurwitz, 1998). These interactions with DNA modifying proteins and cell cycle control proteins suggest that PCNA also plays important roles in other cellular functions such as DNA repair and cell-cycle progression in addition to its roles in DNA replication. The single-stranded DNA binding protein of E. coli is a homotetrameric protein. In human and other eukaryotes it is called replication protein A (RP-A) or replication factor A. RP-A is a heterotrimer of 70 kD, 32-34 kD, and 11-14 kD subunits (Wold and Kelly, 1988; Marton, et al., 1994), and none of three subunits share any sequence homology to E. coli SSB. Interestingly, mitochondrial SSBs are similar to E. coli SSB in sequence and structure (Thommes, et al., 1995). All SSBs show preferred binding affinity to single- 22 stranded DNA over duplex DNA. DNA helicases are enzymes that use the energy of NTP hydrolysis to processively denature duplex DNA to provide single-stranded DNA template for DNA replication. Helicases also play essential roles during DNA repair, recombination and transcription (Lohman, 1993). The DNA unwinding requirement in these processes vary considerably and so do diverse interactions between helicase and other proteins. It is not surprising that many DNA helicases have been purified and characterized and numerous orphan helicases have been isolated either by sequencing projects or by genetic cloning approaches. Only those helicases that have been shown to interact or associate (co-purification) with DNA replication proteins or DNA polymerases are now considered to be required for DNA replication. OVERVIEW The overall sequence similarity between DNA pols from different families is very weak and they show great diversity in their subunit structures and biochemical activities. However, experimental evidence is accumulating from genetic, biochemical and structural studies that supports the hypothesis that family A, B, and C DNA polymerases are all related and share a common ancestor (Zhu and Ito, 1994). One fundamental feature of all DNA polymerases that is absolutely conserved from bacteria to man is the chemical mechanism of the template-primer DNA dependent nucleotidyl transfer reaction. Their substrates in various DNA metabolic pathways vary from a single gap in base excision DNA repair to an entire chromosome of over a 100 million nucleotides in mammalian cells. DNA synthesis in various pathways is also regulated in coordination with other aspects of the life of a particular cell. Thus, the requirements of each specific pathway determine the 23 structural and mechanistic diversity in DNA pols. It is not surprising that DNA polymerases perform basically the same chemical reaction whether in DNA replication or DNA repair but show great diversity in their structures. However, I find it almost amazing that replicative DNA polymerase holoenzymes in different systems have evolved to perform other biochemical functions required for DNA synthesis with non-homologous proteins and even by completely different mechanisms. For example, the complex for high processivity in E. coli DNA replication is a dimer of B subunits in pol III(Kong, et al., 1992), while PCNA used by DNA pols in eukaryotic cells is a homotrimer (Krishna, et al., 1994). The B subunit and PCNA show no sequence similarity yet both proteins fold to nearly superimposable 3- dimensional structures and increase the processivity of DNA pols by a ‘sliding clamp’ mechanism. It is equally interesting that bacteriophage T7 DNA polymerase binds a host cellular protein, thioredoxin, to increase its processivity (Tabor, et al., 1987). The structural data by Doublie et al. (Doublie, etal., 1998) have demonstrated that thioredoxin binds to the tip of the thumb domain of T7 pol and forms a flexible structure which could close the DNA-binding groove of T7 pol. The herpes virus DNA pol might employ the same mechanism although structural data are not available (Hernandez and Lehman, 1990). Another example is the priming of DNA synthesis. The mechanism of elongating established primer chains by all DNA polymerases appears to be absolutely conserved based upon many lines of evidence. However, the processes of starting a new DNA strand are quite diverse. Initiation of chromosomal DNA replication in both bacteria and eukaryotes involves the loading of a primase to the unwound DNA. Again, different strategies are employed to locate the primase to the origin. The primase can be a separate enzyme as in E. coli , or contained in the same protein complex functioning as helicase as in herpes simplex virus (HSV) (Crute and Lehman, 1991), or in complex with a DNA 24 polymerase (pol or) as in eukaryotes (Lehman and Kaguni, 1989). In eukaryotic cells, both nuclear and mitochondrial DNA synthesis are generally primed by short RNA primers, but the enzymatic mechanisms of primer synthesis are different. Pol 0t contains a primase subunit to provide short RNA primers (Kaguni, et al., 1983a; Schneider, et al., 1998). In contrast, leading strand initiation in mtDNA replication uses RNA synthesized by mitochondrial RNA polymerase (Lee and Clayton, 1998). Some viruses have evolved mechanisms that do not even require RNA synthesis; they use either an origin-binding protein (King, et al., 1997; Mendez, et al., 1997) or self-complementary hairpin termini (adeno-associated viruses) to supply the 3' hydroxyl ends (Ward and Bems, 1995). The unique mechanism used by HIV reverse transcriptase is recruitment of a tRNA as the primer. From the results of previous work in our laboratory it is clear that the B subunit of Drosaphila pol y is a distinct protein, not a proteolysis product of the catalytic subunit derived during the purification (Olson, et al., 1995), and that the B subunit binds to the at subunit with high affinity. We have not determined the functional role of the B subunit in biochemical studies. Many protein factors required for mtDNA replication have not been identified, and accessory subunits of different DNA pols can exhibit several biochemical activities and/or structural roles. In fact, the B subunit could perform one or several functions required for efficient and high fidelity mtDNA synthesis. My thesis research aims to determine (1) whether or not the B subunit is a bonefide subunit of the enzyme, (2) the sequence identity of this subunit, (3) whether pol ys from other sources also contain this subunit, and finally (4) the biochemical function of the B subunit in mtDNA 25 replication. The focus of my research is to clone the gene encoding the B subunit, and establish an expression system to produce, purify, and characterize recombinant pol y. The low abundance of native pol y limits the physical amount of enzyme that can be purified, thus prevent us from performing interesting structure-function studies which require a large quantity of protein. The successful expression and purification of recombinant enzyme will not only provide us materials, but also an important methodology to study mutant forms of the enzyme and its individual subunits. 26 CHAPTER II INTERACTION BETWEEN DROSOPHILA POL 7 AND TEMPLATE—PRIMER DNA 27 INTRODUCTION Many enzymes are required to replicate mtDNA completely. However, DNA polymerase 'y (pol y) is the key enzyme in mtDNA synthesis. It has been documented that Drasaphila pol y can utilize efficiently a variety of DNA substrates including gaped DNA, poly(rA).oligo(dT), and singly-primed ¢X174 DNA (Wemette and Kaguni, 1986). Whether the template-primer DNA is presented by an RNA-processing enzyme acting on an RNA transcript created by mtRNA polymerase, or synthesized by a primase activity, specific interactions between pol 'y and the template-primer DNA is a required step in the transition from initiation to processive elongation in both leading and lagging strand DNA replication. The functional interaction of pol y and template-primer DNA is also a good target for the regulation of mtDNA replication, considering the proposed continuous asymmetric model of animal mitochondrial DNA replication. According to this model, for both leading and lagging DNA strand synthesis, pol 7 needs to recognize and interact with the primer terminus only once if processive DNA polymerization is maintained. The large at subunit of pol 7 contains both DNA polymerase and exonuclease activities (Lewis, et al., 1996), and the accessory B subunit is required for efficient catalysis (Olson, et a1 1995). It is not known whether the B subunit is involved in DNA-protein interactions when pol y binds to template-primer DNA. It is also not known whether the B subunit plays different 28 roles in the initiation and elongation of continuous mtDNA synthesis. A number of experimental approaches have been developed to study the specific interactions between DNA-binding proteins and substrate DNAs. Gel mobility shift electrophoresis is a very effective method to probe the DNA sequence specificity and stability of DNA-protein complexes. Many sequence-specific DNA binding proteins have been shown to form covalent DNA-protein complexes when exposed to UV light (Safer, et al., 1988; Capson, et al., 1991). Substituting thymidine monophosphate with 5-bromodeoxyuridine monophosphate in the binding site sequence of a DNA substrate increases the photoreactivity of the DNA and better cross-linking efficiency is obtained (Blatter, et al., 1992). The structural change introduced by this substitution is minimal since 5-bromouracil (BrU) and thymine have very similar van der Waals radii (Blatter, et al., 1992). Most sequence-specific DNA binding proteins also do not distinguish between BrU and thymine so their DNA binding specificity and affinity are not greatly affected. BrU itself is not active in the dark. However, a highly reactive uracilyl radical is generated upon its exposure to UV light. If there is a hydrogen donor in direct contact with the radical, such as when the DNA is bound to a protein, covalent bond formation may occur between them. When long wavelength UV is applied, no DNA strand break or DNA-DNA cross-linking or protein-protein cross-linking is observed (Hockensmith, et al., 1986; Lin and Riggs, 1974). This technique has been applied to identify the catalytic subunit of X. laevis pol y (Insdorf and Bogenhagen, 1989a). Combined with proteolytic mapping, the specific DNA binding domain and even the specific amino acid residues involved can be identified. Enzymes with multiple activities such as DNA polymerases are usually structured as a series of independent functional domains connected by short polypeptide linkers which are more sensitive to proteolytic attack. Partial proteolysis of such proteins can yield active fragments that can define the functions of multiple domains. 29 The DNase I footprinting technique has been applied to study the specific contact between a DNA polymerase and its DNA substrate as well as the interactions between the DNA polymerase and other accessory factors at the template-primer junction. E. coli DNA polymerase HI holoenzyme has been shown to footprint three helical turns on the primer strand. (Reems and McHenry, 1994). T4 DNA polymerase and four other replication proteins interact at the template-primer junction and form different complexes when various complex members are present (Munn and Alberts, 1991). One complication of footprinting with DNA polymerase containing an intrinsic 3'-5' exonuclease activity is that the exonuclease activity will act on the 3'-hydroxyl ends generated by the DNase I digestion, thus destroying the initial footprint, because the DNA polymerase is presented in molar excess in order to form stable DNA-protein complexes. The DNA substrate used must be labeled at the 3' end of the primer strand, so the DNase I fragments with newly-created 3' ends will not be labeled and not observed on the gel. Incorporation of a dideoxynucleotide at the 3' end of the primer strand renders the substrate resistant to hydrolysis by the 3'-5' exonuclease activity of pol 7 because dideoxynucleotides are potent inhibitors of pol 7. To elucidate structure-function relationships in Drasophila pol y, we employed the gel mobility shift assay combined with UV-induced cross-linking, and DNase I footprinting to study the molecular interactions between the pol y holoenzyme and template- primer DNA. In addition, we examined the effect of mitochondrial single-stranded DNA- binding protein (mtSSB) on these interactions. 30 EXPERIMENTAL PROCEDURES Materials Nucleotides and Nucleic Acids- The DNA substrate used for gel electrophoretic mobility shift and photocross-linking experiments is a synthetic 36 mer oligonucleotide (LSK-7, Figure 1C ) which will form a hairpin structure with a 5'-overhang under native conditions, thus creating a primed template for pol 7 action. DNase I footprint substrate DNA is constructed by annealing a SO-mer oligonucleotide complementary to Ml3mp19 single stranded DNA at position 991 to 1041. Both oligonucleotides were synthesized in an Applied Biosystems mode 477 oligonucleotide synthesizer. Ml3mp19 single stranded DNA was prepared by standard laboratory methods. Unlabeled deoxyribonucleoside triphosphates were purchased from P—L Biochemicals. [Ct-32P]dATP was purchased from ICN. Enzymes and Proteins - Drosaphila pol 7 Fraction VI (glycerol gradient fraction) was prepared as described by Wernette and Kaguni (Wernette and Kaguni, 1986). Drasaphila mtSSB was purified from embryonic mitochondria by a variation of the published method [Thommes, 1995 #923 Farr and Kaguni, unpublished data]. Trypsin and DNase I were purchased from Sigma. Chemicals- Protease inhibitor PMSF was purchased from Sigma, sodium metabisulfite was from J. T. Baker Chemical Co., and leupeptin was from Peptide Institute, Minoh-Shi, Japan. 31 Methods Binding and Photochemical Cross-linking of pol yta Template-primer DNA -- A radiolabeled deoxyoligomer (40 nt) containing a 3’-terminal bromodeoxyuridylate residue was prepared by incubating the 36-mer DNA (LSK7, 22 pmol) described under “Materials” with E. coli DNA polymerase I Klenow fragment (0.4 units) for 10 minutes at 37 0C in the presence of BrdUTP (16 pmol) and [ct-32P]dATP (26 pmol), in a reaction mixture (0.2 ml) containing 50 mM Tris‘HCl pH 8.0, 10 mM MgC12, 4 mM dithiothreitol, and 200 [lg/ml bovine serum albumin. The mixture was incubated further for 30 minutes at 37 0C after addition of unlabeled dATP (5 nmol). The reaction was terminated by addition of EDTA to 10 mM, and heating for 15 minutes at 65 0C. The resulting radiolabeled 40-mer was precipitated with ethanol, resuspended in 10 mM TriS'HCl (pH 8.0), 1 mM EDTA, and its length and purity were analyzed by 18% polyacrylarnide gel electrophoresis in the presence of 7 M urea (data not shown). Template-primer DNA binding by pol 7 was examined in a gel electrophoretic mobility shift assay as follows. Pol 7 Fraction VI (28 fmol) was incubated with the [32P]dAMP-labeled BrdUMP-substituted 40-mer (0.22 pmol) in the absence or presence of competitor DNA for 10 min at 30 0C in standard pol 7 reaction buffer containing 50 mM Tris'HCl (pH 8.5), 4 mM MgCl2 , 5 mM dithiothreitol, and 30 mM KCl, followed by the addition of bromphenol blue and glycerol to 0.001 and 5%, respectively, and 32 electrophoresis in a 4.5% native polyacrylamide gel (13x13x.15 cm) in 45 mM Tris-borate (pH 8.3) and 1 mM EDTA. After electrophoresis, the gel was dried under vacuum and exposed at -80 0C to Kodak X-Omat AR X-ray film using a DuPont NEN Quanta III intensifying screen. Photochemical cross-linking with UV light was performed after incubation of pol 7 Fraction V1 with template-primer DNA as described above. The reaction mixtures were irradiated for 15 min at 0 0C with UV light (300 nm) from a germicidal bulb (Fotodyne, 4 X 15 watts) at a distance of 8 cm. After irradiation, the samples were made 1X in Laemmli sample buffer (Laemmli, 1970), denatured, and electrophoresed in a 7.5% SDS- polyacrylamide gel (13x13x0.15 cm). The gel was dried under vacuum and autoradiographed as described above. In experiments where pol 7 was subjected to digestion with trypsin prior to photochemical cross-linking, the DNA binding mixtures contained higher levels of pol y (0.6 pmol), template-primer DNA (1.5 pmol ), and trypsin (200 or 400 ng) and were incubated for 15 min at 20 0C in a digestion buffer (25 pl) containing 40 mM potassium phosphate pH 7.6, 160 mM ammonium sulfate, 0.012% Triton X-100, 1.6 mM EDTA, 5mM DTI‘, 0.8 mM PMSF, 8 mM sodium metabisulfite, 1.6 jig/m1 Leupeptin, and 16 % glycerol. The digestion was terminated by addition of sodium metabisulfite to 20 mM and leupeptin to 20 ug/ml. Aliquots of samples were then added to the DNA binding reaction mixture, processed, irradiated and electrophoresed as described above. The trypsin cleavage products of pol 7 were also resolved on 7.5 % SDS-polyacrylamide gel and identified by silver staining. DNase I Footprinting -— The footprinting substrate DNA was constructed by 33 annealing a 50-mer oligonucleotide complementary to M13mp19 single stranded DNA at position 991 to 1041 in a buffer containing 40 mM Tris pH 7.5, 20 mM MgC12, 50 mM NaCl to the circular single-stranded M13mp19 DNA. The primer strand was then further extended by Sequenase Version 2.0 with [a-32P]dATP (3000 Ci/mmol, 0.13 1.1M) and dd'ITP (0.8 uM). Excess unannealed primer DNA and unincorporated nucleotides were removed by gel filtration on a Sephadex G-50 column equilibrated with 10 mM Tris-HCl pH 8.0, 1 mM EDTA. The DNA concentration of purified template-primer DNA was determined by measuring the absorbance at 260 nm. The specific activity of labeled DNA was determined by liquid scintillation. The final DNA product was also analyzed on 15 % polyacrylamide-urea gel to assess the size of primer strand DNA. The DNase I digestion reaction was performed in a reaction buffer of 50 mM Tris pH 8.5, 4 mM MgC12, 5 mM DTT, 30 mM KCl. The amount of DNase I needed was determined by titration to be 0.25 ng/ul. 17 fmol of DNA was used for all of the reactions, but various amounts of pol y and mtSSB were added in different sets of experiments. After mixing all the reagents except the DNase I, the reaction mixtures were incubated at 37°C for 5 minutes to reach equilibrium. An additional 2 minutes of incubation at 37 °C was performed after the DNase I was added. The digestion was terminated by adding 150 111 of stop buffer (0.5 % SDS, 0.2 M sodium acetate, 30 mM EDTA, 25 ug/ml tRNA). The quenched reaction mixtures were then extracted with an equal volume of phenol/chloroform (1:1), and precipitated with ethanol at - 80 °C for 30 minutes. The DNA pellets were washed with 70 % ethanol and dried in a vacuum desiccator. The DNAs were redissolved in 6 ul of 95 % formamide containing 10 mM NaOH, 1 mM EDTA, and 0.3 mg/ml each of bromophenol blue and xylene cyanol as tracking dyes. Aliquots from each reaction were denatured for 2 min at 100 °C, chilled on ice and electrophoresed in a 10 % polyacrylamide DNA sequencing gel 34 containing 7 M urea in 90 mM Tris-borate pH 8.3 and 2 mM EDTA. Autoradiographs were obtained by exposing the dried gel to Kodak X-Omat AR x-ray film. RESULTS Drosophila pol 7 forms a discrete complex with template-primer DNA Drosophila pol y catalyzes efficient DNA synthesis on a variety of template-primer DNAs (Wernette, et al., 1988). To begin to elucidate the roles of its two subunits in enzyme function, template-primer DNA binding by native pol 7 was examined by gel electrophoretic mobility shift assay and UV-induced cross-linking experiments. A radiolabeled, partially double-stranded oligomer of 40 nt that contains a 3’-terminal bromodeoxyuridylate residue (see Figure 1C) was synthesized and used as the substrate for the DNA binding assay. A single discrete enzyme-DNA complex was observed and the presence of both DNA substrate and Drosophila pol 7 was necessary and sufficient for the formation of this complex ( Figure 1A). The incorporation of BrdU at the 3' end of the primer strand of the template-primer substrate does not affect the recognition and binding by pol 7. Furthermore, the enzyme-DNA complex formed by Drosophila pol 7 was detected over a 10-fold range of pol y to DNA molecules (data not shown), and the stable complex formation could be competed away nearly completely by a 20-fold excess of unlabeled DNA substrate (Figure 1A). 35 Fig. 1 Binding and photochemical cross-linking of Drosophila pol y to template-primer DNA. A, template-primer DNA binding by pol 7. Po] 7 Fraction VI (0.36 units, 28 fmol) was incubated with radiolabeled bromodeoxyuridylate-substituted template-primer DNA (shown in C, 0.22 pmol) for 10 min at 30°C as described under "Methods," and the reaction products were electrophoresed in a 4.5% native polyacrylamide gel and the gel autoradiographed. Lanes 1 and 2 represent no protein and no DNA controls, respectively. Lanes 3-5 represent samples containing both pol y and radiolabeled DNA substrate in the absence (lane 3) or presence of unlabeled competitor DNA (lane 4, 2.2 pmol; lane 5, 4.4 pmol). B, photochemical cross-linking of pol y to template-primer DNA. Pol 7 was incubated with template-primer DNA as in A. The samples were then irradiated with UV light (300 nm) for 15 min at 0°C, processed, and electrophoresed in a 7.5% SDS-polyacrylamide gel, and the gel was autoradiographed as described under "Methods." Lanes 1 and 2 represent no protein and no irradiation controls, respectively. Lanes 3-5 represent irradiated samples containing both pol y and radiolabeled DNA substrate in the in the absence (lane 3) or presence of unlabeled competitor DNA (lane 4, 2.2 pmol; lane 5, 4.4 pmol). C, template-primer DNA. A partially double-stranded deoxyoligomer (40 nt) containing [32P]-dAMP (*) and bromodeoxyuridylate (B in sequence) at its 3'-terminus was prepared as described under "Methods." 36 Binding and photochemical cross-linking of Drosophila pol y to template-primer DNA 1 2 3 4 5 1 2 3 4 A. Gel Mobility Shift B. SDS Denaturing Gel C C ‘Y r r C C v 7 Comp. DNA 10x 20x 10X DNA-pol y Complex A 0 DNA Submte C C CAGGCCGTCAAAB—B' T A GTCCGGCAGTTTAATACCTCT-S' C FIGURE 1 37 20X The catalytic subunit of Drosophila pol 7 makes close contact with template- primer DNA To probe the involvement of the two subunits of Drosophila pol y in template- primer DNA binding, the enzyme-DNA complex was subjected to photocross-linking in the presence of UV light and the covalently cross-linked enzyme-DNA complex was analyzed by denaturing SDS-polyacrylamide gel electrophoresis and autoradiography (Figure 13). The catalytic 0t subunit but not the B subunit in native pol y can be cross-linked to the radiolabeled BrdUMP-substituted template—primer DNA, and the complex can be competed away by 20-fold excess of unlabeled DNA substrate as well (Figure 1B). Control experiments were also performed to show that UV irradiation did not cause DNA-DNA cross-linking, that the enzyme-DNA complex was stably maintained after UV irradiation, and that the formation of cross-linked products was UV dose dependent (data not shown). The catalytic subunit of native pol 7 has a molecular weight of 125 kDa, and the covalently cross-linked enzyme-DNA complex migrated on the denaturing SDS-polyacrylamide gel with an apparent molecular mass of 140 kDa (the calculated molecular weight of substrate DNA is 13 kDa). Both micrococcal nuclease and DNase I could cleave the DNA in the cross-linked complex but yielded products differing in size presumably resulting from the cleavage at different sites on the DNA substrate. Combined action of both nucleases produced a final product very close in size to the molecular weight of the or subunit itself (Figure 2). There was no detectable cross-linking product of 50 kDa, the expected protein- DNA complex if the B subunit alone were cross-linked to the substrate DNA. In addition, we did not detect a cross-linked product of 170 kDa which would be the expected protein- DNA complex if both subunits were covalently cross-linked to the DNA. From the results 38 Fig. 2 Photochemical cross-linking of Drosophila pol y to template-primer DNA covalently links the catalytic subunit to primer DNA. Pol 7 (0.36 units, 28 fmol) was incubated with radiolabeled and BrdUMP-substituted template-primer DNA (0,22 pmol), and irradiated with UV light (300 nm) for 15 min at 0°C, and the DNA- enzyme complex was digested with micrococcal nuclease (laneZ, 1 pg) or DNase I (lane 3 , 2 jig and lane4 10 ug), and electrophoresed in a 7.5% SDS-polyacrylamide gel, and the gel was autoradiographed as described under "Methods." Lanes I represent samples without nuclease digestion. The molecular masses of DNA-protein complexes were determined by the protein standard electrophoresed in the same gel and stained with silver. 39 Catalytic subunit of Drosophila poly is cross-linked DNA (0.2 pmol) pol y (28 frnol) UV DNase 1 Mi. nuclease to the template -primer DNA +++N + ++++w FIGURE 2 40 - 140 kDa - 120 kDa of cross-linking experiments we concluded that the template-primer DNA-binding domain(s) of native pol 'y resides in the or subunit. Meanwhile, this result does not provide conclusion about the role of the B subunit in DNA-binding. However, these results do indicate that either the B subunit does not bind to DNA directly or that it does not make close contact at the template-primer junction of the DNA substrate when native pol y holoenzyme binds to DNA. The recombinant catalytic subunit of Drosophila pol y is active in template- primer DNA binding The same gel electrophoretic mobility shift and UV-induced cross-linking assays were performed using recombinant catalytic subunit of pol 7 expressed and purified from E. coli (Lewis, et al., 1996) to study the DNA-binding activity of the catalytic subunit. In the absence of the B subunit, the catalytic subunit is also able to recognize the DNA substrate and form a stable discrete complex (Figure 3). Further, photochemical cross- linking of the catalytic subunit-DNA complex induced by UV light demonstrates a specific association of the catalytic subunit with the template-primer DNA. (Figure 3) A proteolytic fragment of the catalytic subunit retains DNA binding activity To dissect functional domains in Drosophila pol y, the purified native enzyme was subjected to limited tryptic digestion in the presence of template-primer DNA, followed by UV cross-linking and then denaturing SDS-polyacrylamide gel electrophoresis analysis and autoradiography. Limited tryptic digestion of native pol 'yproduces a form of the 41 Fig. 3 Binding and photochemical cross-linking of the recombinant catalytic subunit of Drosophila pol yto template-primer DNA. A, template-primer DNA binding by the recombinant catalytic subunit of Drosophila pol y. Bacterially expressed catalytic subunit (urea-extracted insoluble fraction, 70% pure) was incubated for 10 min at 30°C with radiolabeled bromodeoxyuridylate-substituted template-primer DNA as described under "Methods," and the reaction products were electrophoresed in a 4.5% native polyacrylamide gel and the gel autoradiographed. Lanes 1 represents a no protein control. Lane 2 represents a sample containing Po] 7 Fraction VI (0.09 units, 7 fmol) and radiolabeled substrate DNA (0.11 pmol). Lanes 3-5 represent samples containing0.06 units (96 fmol) of recombinant catalytic subunit and radiolabeled substrate DNA (0.22 pmol) in the absence (lane 3) and presence of unlabeled competitor DNA (lane 4, 1.1 pmol; lane 5, 2.2 pmol). B, photochemical cross-linking of the recombinant catalytic subunit to template-primer DNA. The samples were incubated with labeled substrate DNA as in A and irradiated with UV light (300 nm) for 15 min at 0°C. The DNA-protein complexes were processed and electrophoresed in a 7.5% SDS-polyacrylamide gel, and the gel was autoradiographed. Protein, labeled substrate DNA, unlabeled competitor DNA were as in A. Lane 1 represents a no protein control. Lane 2 represents a sample containing P01 7 Fraction VI. Lanes 3-5 represent irradiated samples containing the recombinant catalytic subunh. 42 Binding and photochemical cross-linking of the recombinant catalytic subunit of Drosophila pol y to template--primer DNA A l 2 3 4 5 C y a a ct Competitor DNA 5X 10X 0. — DNA Complex FIGURE 3 43 Fig. 4 Photochemical cross-linking of trypsin-digested Drosophila pol yto template-primer DNA. P01 7 Fraction VI (7 units,0.6 pmol) was incubated for 15 min at 20°C with radiolabeled BrdUMP-substituted template-primer DNA (0.2 pmol), in the absence (lane 1 and 2) or presence of 200 ng (lane 3) or 400 ng (lane 4-6) of trypsin, and the digestion was terminated by addition of a large excess of protease inhibitors as described under "Methods." Following incubation, the samples were irradiated with UV light for 15 min at 0°C, processed, and electrophoresed in a 7.5% SDS-polyacrylamide gel, and the gel was autoradiographed. Lane I, a UV-irradiated, no protein control; lane 2, pol y irradiated without prior trypsin digestion; lane 3 and 4, pol y digested with 200 or 400 ng of trypsin, respectively, prior to UV irradiation; lane 5, a UV-irradiated, trypsin only control; lane 6, as in lane 2 except that bovine serum albumin was substituted for pol y. Photochemical cross-linking of trypsin-digested Drosophila pol y to template-primer DNA SDS Denaturing Gel l 2 3 4 5 6 C y y y Tryp. BSA (1 - ,' 0' g o ' T '1. '. Ts " I ' . ' .. ’ p .. Q C sir. . 5. f" ‘ Fuses-i. .1 FIGURE 4 45 enzyme that retains DNA binding activity, at a level that produces a cross-linked product comparable in intensity to the intact enzyme (Figure 4). In the proteolyzed form, the pol y catalytic subunit is trimmed from a 125- to a 65 kDa DNA-binding polypeptide. Staining of the SDS-polyacrylamide gel with silver nitrate identifies two predominant digestion products of the catalytic subunit of 65 and 55 kDa, and an intact B subunit (data not shown). Notably, the same results are obtained when template-primer DNA is added before or after digestion with trypsin. However, the binding of template-primer DNA appears to protect the enzyme from further degradation, while nearly quantitative conversion of the a subunit from a 125- to a 65-kDa DNA-binding polypeptide is observed in the presence of template-primer DNA (Figure 4), it is cleaved further to yield smaller polypeptides in the absence of template-primer DNA, at a point where about 50% of the CL subunit remains intact (data not shown). The data presented here suggest that the 65-kDa polypeptide represents both a structural and a functional domain of the or subunit with respect to DNA binding activity. Whether or not this form of the enzyme exhibits either DNA polymerase or 3’-5’ exonuclease activity or if the B subunit remains associated, remains to be determined. Cross-linked Drosoph ila pol y retains DNA polymerase activity The cross-linked pol y holoenzyme apparently retains DNA polymerase activity as determined by first producing the covalently cross-linked enzyme—DNA complex with unlabeled DNA substrate and then initiating DNA synthesis in the presence of radiolabeled nucleoside triphosphate. A radiolabeled enzyme-DNA complex can be detected on a denaturing SDS-polyacrylamide gel (Figure 5 lane 3) indicating that pol y can add an 46 Fig. 5 Cross-linked Drosophila pol y retains DNA polymerase activity. Po] 7 Fraction VI (0.35units, 0.03 pmol) was incubated for 10 min at 20 0C with BrdUMP- substituted template-primer DNA (0.2 pmol). Following incubation, the samples were irradiated with UV light for 15 min at 0 0C, and further incubated with [32P]-d'ITP (lane 3) at 25 0C for additional 20 min, processed, and electrophoresed in a 7.5% SDS- polyacrylamide gel, and the gel was autoradiographed. Lane 2 represents no [32P]-dTTP control. Lane 1 represents a cross-linked DNA-enzmye complex using radiolabeled DNA substrate as control. 47 Cross-linked Drosophila pol y catalytic subunit retains DNA polymerase activity ' a/DNA FIGURE 5 48 additional nucleoside monophosphate to the 3’ terminus of the primer even when it is covalently linked to the substrate DNA. This result also suggests that at least a significant amount of DNA-pol 7 complex is maintained in a state such that the 3’ end of the primer strand is located in the DNA polymerase active site. D ro s op h ila pol 'y footprints two helical turns of its primer P01 7 holoenzyme purified from Drosophila embryos forms a highly stable complex with template-primer DNA. When enzyme/template—primer DNA complexes were subjected to DNase I footprint analysis, about 20 nucleotides from the 3' end on the primer strand were protected against DNase I digestion. The minimum amount of pol 7 required to observe a stable footprint on 17 fmol of DNA substrate was 135 fmol in the absence of any other DNA replication accessory proteins (Figure 6A). This result indicates that the pol y holoenzyme makes close contact with 20 base pairs of duplex DNA, corresponding to two helical turns. Mitochondrial single-stranded DNA binding protein enhances template- primer DNA binding by pol y Mitochondrial single-stranded DNA-binding protein stimulates pol 7 activity (Williams and Kaguni, 1995). This may occur by enhancing primer recognition and binding, by enhancing formation of stable and/or productive pol 'y : template-primer DNA complexes, and/or by stimulating nucleotide polymerization per se. To evaluate these possible mechanisms of mtSSB stimulation, enzyme/template-primer DNA complexes were subjected to DNase I footprint analysis in the presence of a saturating amount of 49 Fig. 6 Drosophila pol y footprints two helical turns of primer DNA and mitochondrial single-stranded DNA-binding protein enhances primer DNA binding by pol y. Singly-primed M13 DNA was radiolabeled at the 3'-end of the primer strand and a ddTMP was incorporated to produce a 3'-5' exonuclease-resistant template-primer DNA substrate. A, DNase I digestion reactions were performed by incubating 17 fmol of substrate DNA with 7 to 135 fmol of pol 7 fraction VI at 30°C for 5 minutes and incubating in the presence of 0.25 ng/ttl of DNase I at 37 °C for an additional 2 minutes. The reactions were quenched and DNAs were recovered by phenol/chloroform extraction and ethanol precipitation. The footprinting products were analyzed by 10% polyacrylamide-7M urea gel electrophoresis and autoradiography. Lane I and 8 represent no protein controls. Lane 2, 3, 4, 5, 6, and 7 represent samples containing 7, 14, 27, 55, 83, 135 fmol of pol 7 respectively. B, DNase I digestion reactions were performed as in A except saturating amount (15 pmol) of mtSSB was also added in the reaction. Lane I and 8 represent no protein controls. Lane 2 represents a mtSSB only control. Lanes 3, 4, 5, 6, and 7 represent samples containing 7, 14, 27, 55, 83 fmol of pol 7 respectively. 50 Drosophila pol y footprints two helical turns of primer DNA and mtSSB enhances primer DNA binding by pol y A.12345678B.12345678 poly(finol)0 714 27 55 33135 0 o o 714 27 55 83 0 mtSSB(15pmol) + + + + + + ccoogo.-OQI .333332' ‘3” a" S‘si; - w $3855”! is 333.09.. C“! settlers 9; I" "’ ' , , -_'IIO + M is i::;. :. I; 2'22"?" 2 9! OD. -= .. .’.. ;;. : ” FIGURE6 51 mtSSB (15 pmol; 7.5 pmol of mtSSB is required to coat completely the circular M13mp19 single-stranded DNA because each mtSSB monomer binds 17 nucleotides). mtSSB reduced 3- to 4- fold the amount of pol 7 required to observe a stable footprint on the primer terminus (Figure 6B). mtSSB binds single-stranded DNA preferentially, and it did not itself yield an observable footprint on the primer DNA strand. The coating of the single-stranded region of the substrate DNA by mtSSB would eliminate potential non- specific DNA binding by pol 7. When non—specific single-stranded DNA-binding of pol y is greatly reduced by mtSSB, formation of a stable pol 'y/template-primer DNA complex is able to reach equilibrium at a lower concentration of enzyme. DISCUSSION Identification of the molecular interactions that occur between a replicative holoenzyme and the template-primer is a prerequisite for understanding the mechanistic contributions of each subunit of the enzyme and the effects of other replication accessory proteins. Experimental data presented here demonstrate clearly that Drosophila pol y can recognize and bind to template-primer DNA, forming a very stable enzyme—DNA complex at a molar ratio as low as 0.1 pol y molecule/template-primer DNA, without the assistance of any other replication proteins. In contrast, several other DNA polymerases known to associate with accessory proteins for catalytic function, including bacteriophage T4 DNA polymerase (Munn and Alberts, 1991), E. coli DNA polymerase III (Reems and McHenry, 1994), and calf thymus DNA polymerase 8, do not form a stable complex with 52 template-primer DNA in the absence of accessory proteins. That Drosophila pol 7 does so, and catalyzes relatively efficient DNA synthesis on a variety of template-primer DNAs in the absence of accessory proteins (Wernette, et al., 1988), suggests that such factors are not required for mitochondrial DNA replication. However, all of our experiments were performed in vitro and on a model DNA substrate. mtSSB stimulates DNA synthesis by pol y in vitro (Thommes, et a1 1995), raising the possibility that it and potentially other accessory proteins might enhance the enzyme-DNA interactions of pol y in viva, particularly because the concentration of primer termini in mitochondria is much lower. Indeed, the DNase I footprint analysis reveals that mtSSB enhances the specific interaction between pol ‘y and template-primer DNA. In fact, the DNase I footprint analysis of primer binding may represent an underestimate of the effect of increased binding on catalysis because this experimental approach only measures stable enzyme-DNA complexes. mtSSB also exhibits stimulation by additional mechanisms, such as eliminating secondary DNA structures in the template DNA. Photochemical cross-linking of protein-DNA complexes formed with either pol y holoenzyme or recombinant catalytic subunit revealed that the catalytic subunit but not the B subunit makes close contact with the DNA. At the same time, a super-shift assay using subunit-specific antibody in the DNA-binding reaction suggests that the B subunit is physically present in the pol y—DNA complex (data not shown). Furthermore, the recombinant catalytic subunit alone exhibits very similar DNA-binding activity as compared to the native holoenzyme, and limited proteolysis of the complexes with trypsin identified a 65 kDa proteolytic intermediate of the at subunit that retains DNA binding activity. The 65 53 kDa proteolytic intermediate of the at subunit is stabilized by association with DNA during protease digestion. These results suggest that native pol 7 assumes a different conformation when complexed with the DNA substrate and that the accessory subunit is neither required for nor does it affect the DNA binding activity. Bacteriophage T4 DNA polymerase can bind DNA in three different modes. The location of the primer terminus can be either in the DNA polymerase active site, 3’-5’ exonuclease active site, or in an intermediate transition site (Baker and Reha-Krantz, 1998). The exact location of the 3’-end of the primer DNA terminus can not be determined conclusively in our experimental approach, but it is clear that pol y can initiate DNA synthesis efficiently only when the primer terminus is directed to the polymerase active site. We found that the cross-linked pol ‘y holoenzyme retains the ability to add additional nucleotides to the 3’-end of primer strand (Figure 4). This result suggests that the 3’-end of primer strand is oriented in the polymerase active site. mtSSB may facilitate productive template-primer DNA binding and nucleotide polymerization by pol 'y by the combined actions of eliminating non-specific interaction between pol y and single-stranded DNA, eliminating secondary DNA structures in template DNA, promoting DNA-synthesis competent template-primer binding and promoting the transition from initiation to elongation. E. coli Pol III holoenzyme is stimulated by SSB protein, but this stimulation is dependent on the specific protein-protein interaction between the SSB and the x subunit in the 7 complex (Kelman, et al., 1998; Glover and McHenry, 1998). Other DNA polymerases are stimulated by their cognate SSBs and interact with them. For example, T4 gp32 protein and '17 gene 2.5 protein are both SSBs that bind to and stimulate T4 and T7 DNA polymerases respectively. In addition, human SSB (RP-A) 54 has been shown to interact directly with DNA pol 0t. Although we could not determine the biochemical function of the accessory subunit in these experiments, it is possible that the B subunit is required for pol y to interact with other replication proteins including mtSSB. Other possible functions of the B subunit might include mediating the interaction between pol 'y and the RNA primer terminus at mitochondrial DNA replication origins, and conferring processivity upon pol y. FUTURE RESEARCH We have shown previously that the association of the two subunits of Drosophila pol 'y is so strong that the conditions required to physically separate them results in the denaturation of the protein and complete loss of enzymatic activity (Olson, et al., 1995). Here we demonstrated that the catalytic subunit makes close contact with the template- primer DNA. Protein-protein cross—linking reagents can be added to the DNA/pol y/mtSSB complex to detect potential physical interactions between pol y and mtSSB and determine which subunit contacts mtSSB. Whether the accessory subunit contains DNA-binding activity can also be assayed with a recombinant form of the B subunit. We have performed photochemical cross-linking studies with a photo-reactive nucleotide incorporated into the primer strand of a DNA substrate. Interaction between the B subunit and template-primer DNA can also be examined with DNA containing a photo-reactive nucleotide in the template 55 strand or at upstream postitions in the primer strand. When a large quantity of recombinant pol y is available, photochemical cross-linking and limited proteolysis experiments will become very useful to determine the specific DNA binding region of pol y. We performed our footprinting analysis with a labeled primer strand. A footprinting study to observe the overall DNA binding by pol y will require further experiments to determine the protected region on the template strand DNA. We have demonstrated that mtSSB protein enhances the template-primer DNA binding activity of pol y. Footprinting studies can also provide us an assay to probe the effect of other replication proteins in their actions at the replication fork. Reha-Krantz et al. used a base analog, 2-aminopurine, inserted at the 3’ terminus of the primer strand as a fluorescent reporter to analyze the partitioning of primer DNA strands between the exonuclease and polymerase active site of bacteriophage T4 DNA polymerase (Reha-Krantz, et al., 1998, Beechem, et al., 1998). The 3’-5’ exonuclease activity of pol "y is highly mismatch specific (Olson and Kaguni, 1992). We could use this fluorescent reporter substrate to examine the template-primer DNA binding mode of pol y and determine how the mtSSB protein stimulates the polymerase and exonuclease activities of pol y. 56 CHAPTER III MOLECULAR CLONING OF THE ACCESSORY SUBUNIT OF DROSOPHILA POL 'y 57 INTRODUCTION It is now well established that animal mitochondria contain a nuclear-encoded DNA polymerase, which is capable of performing high fidelity mtDNA replication (Kaguni and Olson, 1989) and is very likely also involved in mtDNA repair (Pinz and Bogenhagen, 1998; Longley, et al., 1998a). A second DNA polymerase activity has been reported in yeast mitochondria (Lucas, et al., 1997), and a DNA polymerase bearing sequence homology to pol B has been identified in mitochondria of the protozoan, Crithidia (Torri and Englund, 1995). However, it remains to be determined whether animal mitochondria also contain another DNA polymerase. The genome sequencing projects in progress and subsequent biochemical studies are likely to answer this question in the future, because all of the proteins required for mitochondrial DNA replication are encoded by nuclear genes. D. melanogaster pol y in its native form purified from embryos is a heterodimer of 125 kDa and 35 kDa subunits (W emette and Kaguni, 1986). The large 125 kDa catalytic subunit contains both 5’-3’ DNA polymerase and 3’-5’ exonuclease activities (Lewis, et al., 1996), while no specific biochemical function has been assigned to the 35 kDa subunit. It has been shown that the Drosophila B subunit is a distinct polypeptide and is important to maintain the catalytic efficiency and/or the structural integrity of the holoenzyme (Olson, et al., 1995). The catalytic subunits of pol y from frog, man, mouse, S. cerevisiae, S. pombe, and P. pastoris have also been cloned (Lecrenier, et al., 1997). In one report, pol ypurified from human Hela cells contained a polypeptide of 54 kDa in addition to the catalytic subunit (Gray and Wong, 1992). The purified porcine liver pol ycontains four polypeptides of 120, 55, 50, and 48 kDa (Kunkel and Mosbaugh, 1989), and a preparation 58 of pol ‘yfrom Xenopus laevis embryos possesses several polypeptides of 100, 85, 55, 40, and 31 kDa in association with the 140 kDa catalytic subunit (Insdorf and Bogenhagen, 1989a). Some of these smaller polypeptides are likely proteolytic products of the catalytic subunit that are derived during the purification. Whether pol y is a single polypeptide enzyme, a heterodimer, or has a variable subunit structure among different species remains an unresolved issue. The study presented here documents the molecular cloning of the B subunit of pol y from D. melanogaster, and the subsequent identification of its homologs in man and mouse. The determination of the genomic structure of the Drosophila pol 7 genes and identification of potential transcriptional regulatory elements in the promoter sequence of both genes is also presented. 59 EXPERIMENTAL PROCEDURES Materials Nucleotides and Nucleic Acids- Unlabeled deoxyribonucleoside triphosphates were purchased from P-L Biochemicals. [y-3ZP]ATP was purchased from ICN. Plasmid pUC119, pET-l la, pET-l6b and ltgtll DNAs were prepared by standard laboratory methods. Synthetic oligodeoxynucleotides as indicated in the text were synthesized in an Applied Biosystems model 477 oligonucleotide synthesizer. Enzymes and Proteins- Drosophila pol 7 Fraction VI was prepared as described by Wernette and Kaguni (Wernette and Kaguni, 1986). T4 polynucleotide kinase, T4 DNA ligase and nuclease S1 were purchased from Boehringer Mannheim. Taq DNA polymerase was from GIBCO-BRL. Exonuclease III was from Life Technologies, Inc. Bacterial strains-Ecoli LE392 hst514, hde, supE44, supF58, lac Y1 or (lacIZY)6, galKZ, galTZZ, metBI, trpR55 was used for screening a kgtll ovarian cDNA library from Drosophila melanogaster (the generous gift of Dr. Chuen-Sheue Chiang, Stanford University). E. coli XL-l Blue recAI, endAI, gyrA96, thi, hstI7, supE44, relAI, lac, (F ’proAB, lachZ M15, Tn10(tet')) was used to subclone the B—subunit cDNA for sequence analysis. E. coli BL21 1DE3 ompT, rB' MB‘ was used for the expression of pET-l 1a and pET-l6b (Novagen) constructs. 6O Chemicals- Isopropylthio-BD-galactoside, nitro blue tetrazolium, and 5-bromo-4- chloro-3-indolyl phosphate were purchased from Sigma. Sodium metabisulfite and leupeptin were purchased from the J. T. Baker Chemical Co. and the Peptide Institute, Minoh-Shi, Japan, respectively. Methods Sequence Analysis of D. melanogaster pol ‘y- D.melanagaster pol 'y (255 pmol), purified from 1120 g embryos of average age 9 hours, was processed for peptide sequencing as described by Lewis et a1 (Lewis, et a1, 1996). Protein sequence analysis was performed at Harvard MicroChem (Harvard University). Cloning of the [3 subunit of D. melanogaster pol 7- One of the peptide sequences IVPHDLAEDLNPNDYQAIDIR was used to generate two degenerate oligonucleotide primers for amplifying relevant sequences from a th11 cDNA library derived from D. melanogaster ovarian mRN A by polymerase chain reaction (PCR). The degenerate-sense forward primer coding for the peptide sequence DLAEDL was 5’- GAT(CT)T(CG)GC(ACGT)GA(AG)GA(CI‘)(CT)T-3’. The degenerate-antisense reverse primer corresponding to the peptide sequence YQAIDI was 5’- AT(AG)TC(AGT)AT(ACGT)GC(CI‘)TG(AG)TA-3’. The PCR amplification reaction was performed for 30 cycles of 94°C at 90 s, 50°C for 90 s, and 72°C for 60 s in a reaction volume of 0.1 ml containing 20 mM Tris-HCl, pH 8.4, 50 mM KC1,4 mM MgC12, 1 mM dithiothreitol, 200 11M each dATP, dCTP, dGTP, dTTP, 0.5 pg of kgtll DNA, 50 pmol of forward oligonucleotide primer, and 4 pmol of 5’-32P-labeled reverse oligonucleotide 61 primer (1.1x106 cpm/pmol), and 1.25 units of Taq DNA polymerase. The expected PCR product of 47 bp was obtained, and purified by electrophoresis in a native 15% polyacrylamide gel. The DNA extracted from the gel was subjected to sequencing by the Maxam and Gilbert method (Maxam and Gilbert, 1980). Based on the nucleotide sequence obtained, a 23-nucleotide deoxyoligomer, 5’-AGGACCTCAATCCCAACGACTAC-3’, was synthesized and used to screen the hgtll ovarian cDNA library by the Benton and Davis method (Benton and Davis, 1977) using E. coli LE392 as the bacterial host. Seven positive clones were obtained in the teriary-screen, and one plaque pure isolate with a 1.7-kilobase pair insert was subcloned and sequenced in its entirety on both strands. The insert cDNA was PCR amplified and subcloned into plasmid pUC119 at its unique EcoRI site using E. coli XLl-Blue as the bacterial host. Fourteen nested deletion plasmids were generated using Exonuclease III and S1 nuclease (Henikoff, 1984). A nucleotide sequence of 1569 bp was determined on both strands by automated fluorescent DNA sequencing using the Applied Biosystems Catalyst 800 for Taq cycle sequencing and model 373 DNA Sequencer for the analysis of products. The complete cDNA sequence was assembled using the Sequencher version 2.1.1 software package. Identification of the human and mouse homologs of the [3 subunit of mitochondrial DNA polymerase- Multiple human and mouse EST cDNA sequences were detected from the dbEST (Expressed Sequences Tags) database of the National Center for Biotechnology Information (NCBI) by the BLAST program using the peptide sequence of the B subunit of Drosophila pol y. The partial C-terminal sequence of the putative human and mouse B subunit homologs showed more than 40% identity to the corresponding region of Drosophila protein at the amino acid level. One EST cDNA clone containing the putative 62 human (accession number H05453) and another EST cDNA clone containing the potential mouse B subunit homolog (accession number AA153407) were purchased from Genome System Inc. (8620 Pennell Drive, St. Louis, M063114). The complete DNA sequences of these two clones were determined by automated fluorescent DNA sequencing using the Applied Biosystems Catalyst 800 for Taq cycle sequencing and model 373 DNA Sequencer for the analysis of products. The complete cDNA sequences for both human and mouse homologs were assembled using the Sequencher version 2.1.1 software package. 63 RESULTS Cloning of the B Subunit of Drosophila DNA P01 7 Five amino-terminal sequences were obtained from the purified native B-subunit polypeptide following tryptic digestion and fractionation of the resulting peptides by microbore HPLC (Figure 1). Many attempts were made to screen a hgtll cDNA library derived from D. melanogaster ovarian mRN A (gift of Dr. Chuen-Sheue Chiang, Stanford University) by using degenerate oligonucleotides as probes. However, a large number of false positive clones made this experimental approach impractical (data not shown). Degenerate oligonucleotides corresponding to the amino- and carboxyl-terminal amino acid residues of one of the peptides were constructed, and used to amplify D. melanogaster cDNA fragments by PCR as described under “Methods.” A correctly-sized PCR product was recovered by gel purification, and DNA sequence analysis confirmed that it corresponded to the amino acid sequence of the tryptic peptide. A 23 nt DNA probe was then synthesized, and used to screen a kgtll cDNA library derived from D. melanogaster ovarian mRNA. Tertiary-screen positive clones were obtained at a frequency of 1.5X10'6 phage screened, a frequency comparable to that found in screening the same library for catalytic subunit cDNA clones (5X10‘) (Lewis, et al., 1996). The largest cDNA was 1.7 Kb in length and the nucleotide sequence of clone # 210-1 was determined in its entirety on both DNA strands. DNA sequences of 5’- and 3’- ends of four additional independently- isolated cDNA clones were obtained. All contained an identical 3’- end sequence, and differed only in the length of the 5’-untranslated region (data not shown). Fig. 1 Nucleotide and deduced amino acid sequences of the B subunit of Drosophila DNA polymerase 7. Amino acid residues enclosed in boxes correspond to those for which the amino acid sequence was determined by peptide sequencing as described under "Methods". Nucleotides are numbered from the putative transcriptional initiation site. The putative polyadenylation signal sequence is indicated by a solid line above the nucleotide sequence. (GenBank accession number: U94702) 65 Nucleotide and deduced amino acid sequences of the B subunit of Drosophila DNA polymerase 'y 1 TTTTGTGCTGGTGAGAATGTTGCGTCTGTTGAGTAAACGTTTTTACTGCAAAATTGCAAAAAAATCGAAT 71 GAAAAGGCCACTAAATTGGACTTCAAGCAGCTAACGCATCCCACCAAGGTGCCACAGACACCAGTGGACT 141 CGAAGTTTCCCGACACCAGCGCCTCCGAAATCCAGATCGACACGAAAACCATCCAGTTGCTGGAGCGGCT 211 GTCGCTGGTGGATCTGGATAGCGAACGGGCGCTGGCCACGCTAAAGAGCAGCATACAGTTCGCGGACAAG 281 ATTGCCCATATCAACACGGAGCACGTGCGTCCACTGTACACGGTCCTGGAGCATCAGCGGCTCCAGTTAC 351 GCAACGACCAGGTGACCGAAGGCGATTGCCGGGCGGAGGTGCTCCGGAACGCCAAGGTGACGGACGAGGA 421 CTACTTCGTTTCGCCGCCGGGCAACATTCCGCTGGAGCAATGAGTCGCATACAACGATGCTTTAAGTCCC 1 M S R I Q R C F K S L 491 TGGCTTCCGCTGGCTTCTTTCG$ACCGIGGAAGACAAIAAACIGGAGCIACIGAGICAIGQAAGGGAAIAI 12ASAGFFRTVEDNKLELLSHGREY A 561 GTTGCAACAGCATTGGACACGACTACGTCCCTTGGCTGCGCACCTGGGCGCCACGAGAGAA 35 L Q Q H W T R L R P L A A H L G A T R E 631 CCCATAAATCCAGTCAACATCCAGCGTTTTTCCTTTCCACAAAGCCAGCAATTCCGTAACAATTTCCAAA 58 P I N P V N I Q R F S F P Q S Q Q P R N N P Q K 701 AACTCGTCAAAGATCATCCTCGAAAGGCCAAATGTCCCACTCTACTGAAACATCAGAGCACTTGCTCTGG 82 L V K D H P R K A K C P T L L K H Q S T C S G 771 TCCCACTAGCCATTCCCTATTTGCGATAAA?GGICCIACGCIACAIIIAACCACGGACIICCIAGIGGAAI 105 P T S H S L F A I K G LP T L H, L T T In F L V IE 841 GCCCTGGAGCACTTCTACAACATGCAGCGCGAGAGCAAGATCTGGTGGATGCGTCTTTCCT 128 A L E H F Y N M Q R E S K I W W M R L S S 911 CGAATCCCAGTCGCTACCGiAICGIACCIIGCQAIIIGGCIGAGGAICICAAICCCAACGACIAICAAGfi 152 NPSRYRIVPCDLAEQLNWYOA H 981 ; s :. s s i s . h : i s i s .1; 175 I D I R T S Y G D A G E V 4'- k.. .;.;;k:. k.k;;:; AVEQLSLVRI 1051 GTGGACGACAAAGACTTCCGGCTGCCCGATGCGCGAACGGGTGAAACTGTGCAACCGACTGTCATAAGGT 198 V D D K D F R L P D A R T G E T V Q P T V I R S 1121 CTGTGATAGAGCTGGAAACTACCACCTGCGCTCTGCTTCTGGATGGCTGTGATCATGGCCGGGACTCGCA 222 V I E L E T T T C A L L L D G C D H G R D S Q 1191 ATCCCTGCTCTTACACCGCGTTCTGGCGCCATATCAATGTGGAATTGCCTGTGTGGAAAGTGATTCCGAG 245 S L L L H R V L A P Y Q C G I A C V E S D S E 1261 CTATCTGCTGACTTGTCTGATCTTTGTCAGCATCTGAAACATGTTCTCAACCACGCCGGTCTTAGACTTT 268 L S A D L S D L C Q H L K H V L N H A G L R L S 1331 CTGAAGGGGATGGAATTCGTACGACGAAGAACGCTTCTCATTTAGCAGAGCACTTGCTGGAAACGGATAT 292 E G D G I R T T K N A S H L A E H L L E T D M 1401 GCTGGGCATACCTTATACACTTGTTATAAACGAGCAAACGCTAAGAAACGGACTGATGCAGTTGCGCAGC 315 L G I P Y T L V I N E Q T L R N G L M Q L R S 338 R D T R L A E T I A . I H I S 1521 ACTAAAAAAAAAAAAAAAAAACCGAATTC 'k FIGURE 1 66 An open reading frame of 1083 nucleotides was identified which encodes the B- subunit polypeptide of 361 amino acid residues with a predicted molecular mass of 41 kDa. This is consistent with the size of 35 kDa determined for the small subunit in purified native pol y. The amino-terminal peptide sequences of all five tryptic peptides derived from the B subunit of the purified native enzyme are located within the deduced animo acid sequence of the B-subunit cDNA. However, there are two amino acid residues showing conflicting sequences between the deduced amino acid sequence and the amino acid sequence obtained from the purified native B subunit (Figure 1). There is no other in-frame ATG codon between the 5' end of cDNA and the translational initiation codon, but multiple in-frame stop codons are present in the 5’ untranslated region. The amino-terminal sequence of the mature B subunit was not determined experimentally because of the limited amount of B- subunit polypeptide derived from native pol 7. However, because the amino terminus of one of the tryptic peptides is located at position +19 in the deduced amino acid sequence, the mitochondrial presequence peptide of B subunit is no longer than 18 amino acid residues. This is comparable with the mitochondrial presequence peptide of the or subunit, which was determined experimentally to be 9 residues (Lewis, et al., 1996). To investigate further the discrepancy between the peptide sequence and deduced amino acid sequence of the B-subunit cDNA, DNA sequences from nt # 950 to 1250 were also determined for all seven B-subunit cDNA clones, and the entire cDNA sequence of clone # 211-5 was determined on both strands. Comparison of cDNA and genomic sequence demonstrates that two classes of cDNA clones are isolated and they all represent 67 normal transcripts from the B-subunit gene. These two classes of cDNAs all encode the 35 kDa subunit with substitutions in residue # 188 and 213 in the deduced amino acid sequences. The first class is represented by clone # 210-1, and the amino acid residues at # 188 and 213 are A and T respectively. The second class is represented by clone # 211-5, and the amino acid residues at # 188 and 213 are T and I respectively. The genomic DNA sequence provided by the Berkeley Drosophila Genome Project (BDGP, http://www.fruitfly.edu) matches well with the cDNA clones represented by #211-5. This may reflect the normal polymorphism among biologically functional alleles. Identification of human and mouse homologs of the B subunit of Drosophila pol 7 No homolog of the B subunit of Drosophila pol 7 was identified upon extensive searching of the latest release of the GenBank and EMBL databases (releases 107 and 57 respectively), or the Saccharomyces Genome Database. However, multiple human, mouse, and rat partial cDNA sequences were detected in the dbEST (Expressed Sequences Tags) database of the National Center for Biotechnology Information (NCBI) using the BLAST searching program with the deduced amino acid sequence of the B subunit of the Drosophila pol yas query. For determining both human and mouse homolog DNA sequences, we obtained EST clones (Genome Systems Inc. 8620 Pennell Drive, St. Louis, M063114) containing possible human and mouse homologs respectively. The human cDNA sequence is 1594 bp in length and contains a complete open reading frame. The human B subunit homolog has a deduced amino acid sequence of 372 residues (Figure 2), with a calculated molecular mass of 42.4 kDa. The mouse cDNA sequence is only 1127 68 Fig. 2 Sequence alignment of the B subunit of Drosophila pol y and its mammlian homologs. Amino acid residues that are conserved and similar are represented in uppercase. For mouse and rat sequences, only the C-terminal partial cDNA sequences have been determined. Human and mouse sequences have been deposited in GenBank and their accession numbers are U94703 and AF006072 respectively. The rat sequence is deduced from the EST cDNA sequence obtained from GenBank dbEST database (accession number AA892950). 69 Sequence alignment of the B subunit of Drosophila Dm Hs Mm Rn CONSENSUS Dm Hs Mm Rn CONSENSUS Dm HS Mm Rn CONSENSUS Dm Hs Mm Rn CONSENSUS Dm HS Mm Rn CONSENSUS Dm Hs Mm Rn CONSENSUS Dm Hs Mm Rn CONSENSUS Dm Hs Mm Rn CONSENSUS pol 'y and its mammalian homologs msriq mvdlgggvhg rchslAsag avavdAlhh ffrtVe..Dn kpslepgDs ------------ F-—-A-—- -------—D- erpLAAth K.eqLVAfLe ——--L—A-L- ptleHquc IGVCFHPVfd IGVCFHPVSn IGVCFHPV-- eskIWWles HRLqWWRKFA HRLLWWRKFA HRL-WWRKFA EleLvrivd DhELLHmYPG DQELLHtYPG D-ELLH-YPG DhgrDS...q QLtENSFtRK QLaENSFaRK QL-ENSF-RK anhaGlrLS ELLENG..IS ELLENG..IS ELLENG..Ia ELLENG--IS GLMQLRSRDT GLIHLRSRDT GLIQLRSRDT GLIQLRSRDT GLIQLRSRDT atrepiann nvlktsgkLr SngshSLfa Tinrnngs SnQTpsSVtr S—QT--SV-- snPSRYrivp MSPSNFSSSD MSPSNFSSaD MSPSNFSS-D DkdfrlpdaR NVSkLhG..R NVStIqG..R NVS-—-G--R lelHRVLap KnLhRKVLKL KquRKVLKL K-L-RKVLKL egdGirtTkn VWPGYLETmQ VWPGYSEth VWPGYLETaQ VWPGYLET-Q rLaEtIHISd TMKEMMHISK TMKEMMHISK TMKEMMHISR TMKEMMHISK iQrfsqusQ .EnllhgaLE LE -------- LE Ika.Tlh1t I.GEKTEASL V.GEKTEASL I-GEKTEASL CD1aEdln.p CQDEEGRKGT CQDElGRKGS CQDEEGRKG- tGet.VthV DGRKNVVPCV DGRKNVVPCV DGRKNVVPCV quGI.AcVE HPCLAPIKVA HPCLAPIKVA HPCLAPIKVA HPCLAPIKVA aShlAEHLle SS..LEQLYS SS..LEQLhS SS..LEQLYS SS--LEQLYS VpDYLLnifk LKDFLIKYIS LRDFLVKYLA LRDFLVKYLA LRDFLVKYLA IHCHJRIEZ 7O kleLLShgre aerVSaetl ---L-S---- qFranquV HYV.NCLDLV HYV.NCLDLV HYV-NCLDLV tdFlvePhRA VWFT..PpRT VWFT..PtRT VWFT--P-RT Ndanidirt thtiFPWGK leySFPWGK N-Y--FPWGK IrSV.IELEt L.SVnGDLDr L.SVsGDVDl L-SV-GDLD- stel..SAD LDVGRGPTLE LDVGKGPTVE LDVGKGPTVE LDVGKGPTVE t.DmLGIPYT KYDEMSILFT KYDEMSVLFS KYDEMSILFT KYDEMSILFT n* SAkNV* SASNV* SAgkA* SA-NV- yakquqth reiqukelS ---L ______ .thPrkAkc NKRLPYGLAQ NRKLPFGLAQ NK-LP-GLAQ 1ethqu.R SnQWlDFWLR SsQWsDFWLR S-QW-DFWLR syGDaGevAV ELIETLWNLG EPIETLWNLG E-IETLWNLG ttcAlLngc GmLAYLYDSF GtLAYLYDSF GTLAYLYDSF LSDLCQthh LRQVCQGLfN LRQVCQGLLN LRQVCQGLLN LRQVCQGLLN LVInEqTLrN VLVTETTLEN VLVTETTLEN VLITETTLES VL-TETTLEN bp in length and represents the C-terminal region of the gene; it contains an open reading frame with a deduced amino acid sequence of 296 residues (Figure 2). The mouse B—subunit homolog likely has a similar primary structure because of its high sequence identity (81%) with the human homolog, although its N-terrninal sequence remains to be determined. Alignment of the complete sequences of Drosophila and human, and partial sequences of mouse and rat using the Gap program of the GCG program package (version 7, Genetics Computer Group) show 30% amino acid sequence identity and 60% sequence similarity overall. The C-terminal region is the most conserved of all three proteins. Two putative leucine zipper motifs of the sequence L(X)6L(X)6L(X)6I and L(X)6L(X)6L(X)6L (D rosophila, residue #38 - 59, #321 - 342) and L(X)6L(X)6L(X)6I and V(X)6L(X)6L(X)6M (human, residue #77 - 98, # 328 - 349) are identified at the N- and C-terminal region of the Drosophila and human sequences (Figure 3). The leucine zipper motifs in the mouse sequence are almost identical to the human sequence with only one or two conserved amino acid residue substitutions in each motif (Figure 3). Further, the Drosophila B subunit contains a zinc finger motif of the C2H2 class located between the two leucine zipper motifs. Although the human and mouse B subunits lack this zinc finger motif, they contain a zinc-binding signature sequence (LGDHELLHNMY) found in the zinc-dependent neutral metallopeptidase family proteins (Murphy, et al., 1991). Another notable feature of both the fly and human proteins is that the C-terminal regions are very leucine rich: 18% and 17%, respectively, of the 120 C-terrninal residues are leucines. Protein structure prediction was performed by the BENCHMARK method (http://www.mbi.ucla.edu/people/fischer/BENCH/benchmarkl .html). The fly and human proteins were shown to have similarity with tRNA synthetases based on predicted 71 Fig. 3 Putative structural features of the B subunit of Drosophila pol y and its human, mouse, and rat homologs. The leucine zipper motifs in the Drosophila and mammalian sequences are located in the homologous region at the N- and C-termini. Also identified are a zinc finger motif in the Drosophila sequence and a zinc-binding signature sequence (ZBS) in the human and mouse sequences. 72 Putative structural features of the B subunit of Drosophila pol y and its human, mouse, and rat homologs Dm l—m l’///1 mm 1 HS 1 m [1111] 1188888 I Mm uan will m—‘l Rr ---------------------------- Em: Zinc finger C(X)3C(X)19H(X)3H [IIlIll ZBS Hs LGDHELLHMY Mm LGDQELLHTY m Leu Zipper 1 Dm L(X)6L(X)6L(X)6I Hs & Mm L(X)6L(X)6L(X)6I m Leu Zipper 2 Dm L(X)6L(X)6L(X)6L HS & Mm &Rr V(X)6L(X)6L(X)6M " ‘ "' Not determined FIGURE 3 73 secondary structures. The Drosophila B subunit is also predicted to contain a three-dimensional fold found in moloney murine leukemia virus reverse transcriptase while the human B-subunit protein is predicted to resemble part of HIV reverse transcriptase. These predictions are potentially significant because all the proteins described above are involved in enzymatic interactions with RNA or DNA molecules. Genomic DNA structure of the Drosophila B subunit gene Two genomic sequences containing the B-subunit gene were deposited into the GenBank by the Drosophila Genome center at UC, Berkeley (accession # L39626 and L39627). These two genomic sequences are from a P1 genomic clone (DSOO941) which is mapped to the left arm of chromosome 2 (subdivision 34D2-34D7) near the region containing alcohol dehydrogenase gene (subdivision 35B). Remarkably, the same P1 clone contains the catalytic-subunit gene of Drosophila pol 7 (Lewis, et al., 1996). Further, the genes encoding the two subunits of native pol yholoenzyme are separated only by an interval sequence of 3.8 kilobase pairs, with the B-subunit gene 5'- to the or-subunit gene (Figure 4). Comparison of the genomic and cDNA sequences indicates that the B-subunit gene contains one small intron of 54 nt, that splits the codon specifying amino acid residue A231 in the deduced amino acid sequence. The intron/exon boundaries are GTAAGT (donor site) and TAG (acceptor site) respectively, consistent with the GT ...... AG rule of intron/exon border sequence. The coding sequence of the exon in the genomic clones is 99% identical to that of the corresponding regions of the cDNA. There are two single 74 Fig. 4 Genomic DNA structure of Drosophila pol 7 genes. The organization and structure of the Drosophila pol 7 genes are represented schematically. The boxes represent the exons: closed boxes represent the protein-coding sequences, and open boxes represent either 5'- or 3'- untranslated sequences. The thick line represents the introns and intragenic sequences. Genomic sequences upstream of the putative transcriptional initiation sites of both genes are enlarged to indicate locations of potential DNA-binding sites for transcription factors. The numbers indicate nucleotide positions relative to putative transcriptional initiation sites. KB, kilobase pair. 75 Genomic DNA structure of Drosophila pol 7 genes B Subunit a Subunit 1 KB GRAB] E133- IEZI‘INREERFERFI -807 -295 -230 -2028 —899 -860 494 -53 1 KB FIGURE 4 76 nucleotide deletions in the genomic sequence that disrupt the reading frame, and 11 mismatches between the genomic and cDNA sequences represented by clone # 210-1. Of the latter, only 5 of those mismatches would result in amino acid residue substitutions. Interestingly, the deduced amino acid sequence of the second class of B-subunit cDNA represented by clone # 211-5 matched well with that of the genomic sequence. Thus, the differences between them may well represent normal genetic polymorphism. An additional ORF encoding a protein with predicted molecular mass of 42 kDa is identified in the genomic sequence between the B-subunit gene and the Ot-subunit gene. This gene may also encode a mitochondrial protein ( Dr. Rafael Garesse, Departmento de Bioquimica, Facultad de Medicina, Universidad Autonoma de Madrid, Spain, unpublished results). A search for RNA polymerase 11 promoter elements in the genomic DNA sequence in the region upstream of the putative transcription initiation site (5’- end of the longest cDNA sequence) did not reveal a consensus TATA sequence. A sequence matching the consensus transcriptional initiator TCAGT (Cherbas and Cherbas, 1993) is located in the genomic sequence 16 bp upstream of the 5’- end of the cDNA sequence, and the translational initiation ATG codon is located at a position 459 bp downstream. (Figure 1) Two additional consensus transcriptional initiator sequences are located within 500 bp in the upstream region. Interestingly, a Drosophila promoter-activating element, DRE (DNA replication-related element), previously identified in a number of genes involved in nuclear DNA replication (Matsukage, et al., 1995), is located 230 bp upstream of the putative transcriptional starting site (Figure 4). Further, potential binding sites for nuclear respiratory factor 1 (NRF-l) (Virbasius, et al., 1993) and transcription factor E2F (Ohtani and Nevins, 1994) are located at position -807 and -295, respectively (Figure 4). Notably, consensus DRE element (position -2028), and potential NRF-l (positions -860, 494, -53) 77 and E2F (position -899) binding sites, are also present in the upstream region of the catalytic subunit gene of Drosophila pol 7 (Figure 4 ). The 3'- end of the B-subunit cDNA clone contains an 18 nt poly (A) sequence. Surprisingly, the poly (A) sequence begins within the translational termination codon, TAA (Figure 1). Based on the comparison of the cDNA and genomic sequences, the polyadenylation site is located no more than six nucleotides downstream from the termination codon. This feature is common in mitochondrially-encoded genes, and may be of evolutionary significance. As with the catalytic subunit cDNA, no match to the consensus poly(A) signal sequence (AATAAA) is present in the B-subunit cDNA, although 23/24 of the nucleotides immediately upsUeam of the termination codon are deoxyadenylate or thymidylate residues. Interestingly, both the catalytic- and B—subunit cDNAs contain the sequence of AATATA (at positions -51 and -16, respectively, relative to the polyadenylation site), which might serve as the poly(A) signal. This alternative poly(A) signal has been implicated in directing polyadenylation of mRNAs of Drosophila cyclin A (Lehner and 0'Farrell, 1989), bicoid (Berleth, et al., 1988), orb (0018 RNA-binding protein) (Lantz, et al., 1992), and ch (Polar granule component) genes (N akamura, et al., 1996). The expression of these genes have been shown to be developmentally regulated. Taken together, the presence and locations of common transcriptional and post- transcriptional regulatory signals in the catalytic and accessory subunit genes of Drosophila pol 7 suggest the possibility of coordinate gene regulation of the heterodimeric mitochondrial DNA polymerase. 78 DISCUSSION The redundant nature of mitochondrial DNA does not eliminate the requirement for high fidelity DNA replication, because the accumulation of mutations in mtDNA is linked to many degenerative diseases in humans (Wallace, 1995a). The most important replicative enzyme involved in mtDNA replication is pol 'y. Drosophila pol ‘y (W emette and Kaguni, 1986) and pol ‘yfrom many species including yeast (Foury, 1989) , frog (Insdorf and Bogenhagen, 1989b), chick, pig (Kunkel and Mosbaugh, 1989), and human (Gray and Wong, 1992) have been demonstrated to contain dual enzymatic activities. They possess both highly accurate DNA polymerase and mispair-specific 3’ - 5’ exonuclease activities. While we have shown previously that Drosophila pol y is a heterodimer of 125 kDa and 35kDa subunits (Olson, et al., 1995), the structure of other mitochondrial DNA polymerases remains a controversial issue. In a previous report, physical and immunological evidence had demonstrated clearly that the 35kDa B subunit of Drosophila pol y is structurally distinct from the 125 kDa catalytic subunit (Olson, et al., 1995). The experimental data presented here demonstrate that the B subunit is encoded by a novel nuclear gene, and bears no similarity to the catalytic subunit in its amino acid sequence. Further, subunit-specific rabbit antisera directed against recombinant proteins recognize specifically and immunoprecipitate the native enzyme (Wang, et al., 1997b). Antibodies raised against native pol 7 also recognize the E. coli expressed recombinant B subunit (Wang, et al., 1997b). We conclude that the 35 kDa polypeptide is a bona fide subunit of the Drosophila pol y. 79 The nuclear genes encoding both the catalytic and accessory subunits are mapped to the same genomic region on the left arm of chromosome 2, and they are closely linked with a physical distance of 3.8 kb. This close linkage may be of evolutionary significance, given the current hypotheses regarding the bacterial origin of mitochondria as a cellular organelle. The physical location of these two genes may also provide advantages for the regulation of pol 7 gene expression. The genomic location, similar gene structure ( i.e. TATA-less promoter, presence of consensus transcriptional initiator and alternative poly(A) signal sequences, small introns and short 3’- untranslated region), and the presence of the DRE elements and potential binding sites for transcription factors NRF-l and E2F in the 5’ untranslated regions of both genes, suggest that the two genes share common regulatory mechanisms, and the possibility that they are coordinately regulated. The DRE element is a recognition sequence for an 80 kDa DNA-binding protein, DREF (Hirose, et al., 1993), and is responsible for activating promoters of nuclear replication genes in Drosophila, including PCNA (proliferating cell nuclear antigen), cyclin A and DNA polymerase Ot- encoding genes (Ohno, et al., 1996). Functional NRF-l sites have been identified in many mammalian nuclear genes that encode mitochondrial proteins. These include cytochrome c, mitochondrial transcription factor A, and at least one subunit of each of the respiratory complexes III, IV, and V (Virbasius, et al., 1993; Virbasius and Scarpulla, 1994). The E2F transcription factor was first identified as a cellular DNA-binding activity responsible for activation of the adenoviral E2A gene promoter. Eight members of E2F family in mammals have been shown to form heterodimeric complexes that are crucial for transcriptional activation of genes regulating S phase entry (C-myc and cyclin E) and genes functioning to engage in DNA synthesis (dihydrofolate reductase, thymidine kinase, PCN A and DNA pol 0t) (Zwicker and Muller, 1997). Activity of E2Fs as a transcription activator 80 or repressor is regulated by their interaction with other cell cycle controlling proteins including Retinoblastoma (Rb) tumor suppressor, other Rb family proteins (p107, p130), and cyclin-dependent kinase. Drosophila E2F-1 is essential for activating DNA pol 0t gene expression, and is required for the G1 to S phase transition during embryogenesis (Duronio, et al., 1995). Further, Sawado etal have recently demonstrated that the DREF transcription factor also regulates the expression of the E2F gene in Drosophila (Sawado, et al., 1998). The promoter region of the Drosophila mtSSB gene also contains DRE elements, and these potential binding sites of transcription factor DREF have been shown to be required for promoter activity in an in vivo transfection assay (1. Ruiz de Mena and LS. Kaguni, unpublished results). That the DRE, NRF-l and E2F sequence elements are present in the Drosophila pol 7 genes is consistent with the fact that mitochondrial DNA polymerase is required for mtDNA replication and hence for cell proliferation, and that pol 7 activity varies greatly during development, and is highest in embryos (Wernette and Kaguni, 1986). Molecular cloning, bacterial overexpression and biochemical analysis of the catalytic subunit has allowed the precise assignment of both 5’ - 3’ DNA polymerase and 3’ -5’ exonuclease functions to the 125 kDa subunit of Drosophila pol 7 (Lewis, et al., 1996). Likewise, Foury has shown via its overexpression in mitochondrial extracts, that the 140 kDa polypeptide of the MIPl gene contains both enzymatic activities in S. cerevisiae pol y (Foury and Vanderstraeten, 1992). Graves et al. (Graves, et al., 1998) and (Longley, et al., 1998b) have also shown that the recombinant 140 kDa subunit of human pol yexhibits both activities when overexpressed in baculovirus infected insect cells. Cloning and sequence alignments have demonstrated that a large polypeptide of 115- 140 kDa contains 81 both the conserved DNA polymerase and exonuclease domains in pol y of several yeasts, Xenopus, and mouse (Lecrenier, et al., 1997). However, pol y purified from Xenopus embryos, pig, and human Hela cells has been shown to contain a large catalytic subunit co-purified with several smaller polypeptides, leaving it difficult to determine the native structure of these enzymes. The identification of human and mouse homologs of the B subunit of Drosophila pol 7 suggests that animal mitochondrial DNA polymerases may share a common subunit structure. The accessory subunit of Drosophila pol y and its mammalian homologs represent novel proteins of 41 and 43 kDa, respectively. They both contain two conserved leucine zipper motif sequences. The catalytic subunits of Drosophila , Xenopus, human and mouse pol 7 also contain leucine zipper motifs located between the conserved DNA polymerase and exonuclease domains (Ye, etal., 1996). One class of zinc finger domain has been shown to participate in protein-protein interactions. It is also possible that the putative zinc finger domain in the B subunit may play significant role in its association with the catalytic subunit. We suggest that the putative leucine zipper domains in both subunits participate in subunit interactions because of the genetic, biochemical and structural evidence for an interaction function in other leucine zipper-containing proteins. If so, we would also propose that both the heterodimeric composition and the structural basis for subunit interaction are conserved among animal pol ‘yholoenzymes. Data base searching against the complete yeast genome did not identify any open reading frame with significant homology to the B subunit of D. melanogaster pol 7. However, this does not exclude the existence of a yeast homolog of the B subunit. It is 82 possible that the product of an analogous gene in yeast serves the biochemical function of the B subunit. The yeast MIPl gene encodes a polypeptide of 140 kDa, but it has 200 amino acid residues at its C-terminus that are absent in all other pol y catalytic subunits. Further, the less highly conserved region between the DNA polymerase and exonuclease domains is substantially shorter. If the C-terminal domain of yeast enzyme serves the same function as the B subunit of Drosophila enzyme, then yeast may not require a B subunit. The molecular cloning of the B subunit of D. melanogaster pol y and its mammalian homologs does not suggest a biochemical function for the accessory subunit because it represents a novel gene. However, other DNA polymerases also contain additional subunits to increase the processivity of the enzyme, or to mediate protein-protein interactions between the polymerase and other replication proteins, thereby enhancing the overall efficiency and/or accuracy of DNA synthesis. For example, T7 DNA polymerase recruits E. coli thioredoxin as a processivity factor. The herpes simplex virus UL42 protein functions as a processivity factor for the viral-encoded herpes DNA polymerase. Human pol 8 contains a 50 kDa subunit which is responsible for interaction with PCNA to increase processivity. The newest member of yeast DNA polymerase (pol C) is a two subunit enzyme. Gene Rev3 encodes the catalytic subunit while gene Rev7 encodes a smaller subunit. The association of Rev7 protein with Rev3 protein increases the overall enzymatic activity of pol C (Nelson, et al. , 1996). The protein-protein interaction domain of T7 DNA polymerase recently has been identified between the exonuclease domain and the DNA polymerase domain. We are currently investigating the function(s) of the B subunit of D. melanogaster pol y. We propose that the B subunit of D. melanogaster pol 83 7 could serve a similar function(s) as the smaller subunits in other DNA polymerases discussed above. 84 FUTURE RESEARCH The availability of both cDNA and genomic sequences for both subunits provides us opportunities to explore not only the functions of the individual subunits but also the regulation of pol 7 gene expression. Studies of the recombinant forms of pol yholoenzyme with either wild type sequence or with mutants containing site-specific mutations in specific regions will define the roles played by conserved sequences. The biochemical functions of the accessory subunit can also be elucidated by comparing the biochemical properties of the holoenzyme with those of the catalytic subunit. Other possible roles of pol 7, such as in mtDN A repair can also be tested with model DNA substrates using purified recombinant pol y. Overproduction of recombinant pol y will allow us to apply protein affinity chromatography to identify other mitochondrial replication factors in addition to allowing further structure-function experiments. The functional significance of potential transcription factor binding sites located in the promoters of both genes can be studied by both in vivo and in vitro approaches. The Drosophila DRE-binding factor (DREF), and E2F/DP transcription factor have been identified and cloned. Gel mobility shift assay and DNase I footprinting using recombinant DREF and E2F/DP transcription factors will determine whether these transcription factors recognize the DRE element sequences and E2F binding sites in the promoter region of the pol 7 genes. We can also design in vivo experiments to test whether promoter sequences containing these transcription factor binding sites will activate reporter gene expression in cultured Drosophila cells. We will also take advantage of the excellent genetic methods available in Drosophila to study the tissue and developmental specific effects of mutations in pol 7 genes by 85 creating transgenic flies overexpressing additional copies of pol 'y gene(s). In summary, identification of both cDNA and genomic clones of the pol 7 genes will extend greatly our ability to perform biochemical and genetic studies to further define the critical functions of pol y. 86 CHAPTER IV PRODUCTION AND BIOCHEMICAL CHARACTERIZATION OF RECOMBINANT DROSOPHILA POL y a I'm 87 INTRODUCTION Recent progress in genetic, biochemical and structural studies of DNA-dependent DNA polymerases has expanded our understanding of their structure and mechanism. At least six distinct DNA polymerases have been identified in eukaryotic cells and they are all encoded by nuclear genes (Wang, 1991). A human gene that is homologous to the Drosophila mus 308 gene and potentially encodes a seventh DNA pol (pol 1]) (Harris, et al., 1996), has been recently cloned (GenBank accession number AF052573, Sharief,F.S., Ropp,P.A., and Copeland,W.C.). All DNA polymerases perform the same enzymatic reaction of adding a single nucleotide to the 3’-end of the primer. Eukaryotic cells have evolved a whole set of DNA polymerases to carry out the same DNA synthetic reaction on different substrates in replication, recombination, or repair. However, pol y is the only DNA pol involved in the replication of animal mitochondrial DNA, and it has been suggested to also carry out base excision DNA repair because it contains a 5’-deoxyribose phosphate lyase activity in the catalytic subunit and participates in DNA repair at abasic sites in vitro (Pinz and Bogenhagen, 1998; Longley, et al., 1998a). DNA pols 0t, B, 8, e, and Q have been demonstrated to be nuclear polymerases and involved either in nuclear genome DNA replication or DNA repair. Each eukaryotic DNA polymerase has evolved structural features for specific functions in diverse DNA synthesis pathways. Pol at is exclusively required for the initiation of nuclear chromosomal DNA replication and the priming of Okazaki fragments during lagging strand synthesis, so it also possesses a primase activity. Pol 0t purified 88 from different sources consists of four subunits in every organism examined. The role of pol B in base-excision repair is well established in mammalian systems and it is conserved as a single polypeptide enzyme from yeast to man. However, there is a discrepancy in the subunit structure of pol 8 and 8, despite accumulating evidence that they likely perform the same function in various systems. P01 8 purified from mammalian cells is a heterodimer, but budding yeast Saccharomyces cerevisiae pol 8 may contain a third subunit (Burgers and Gerik, 1998). Pol 8 purified from the fission yeast Schizosaccharomyces pombe contains five subunits (Zuo, et al., 1997). P01 8 derived from human cells has been shown to contain only two subunits of 255 and 55 kDa, while budding yeast pol 8 contains four polypeptides. It is also not known whether the subunit structure of pol 'y is conserved. Pol yfrom Drosophila is a heterodimer of 125 and 35 kDa subunits. In budding yeast, the MIPl gene has been shown to encode the catalytic subunit of pol y and no homolog of Drosophila B subunit has been identified. P01 7 catalytic subunit from human and other mammalian sources also co-purifies with smaller polypeptides. Whether one of these small polypeptides with apparent molecular mass of 40 to 50 kDa is the gene product of Drosophila B«subunit homolog in mammals remains to be determined. If the Drosophila B- subunit homolog in mammals does encode an accessory subunit of pol 7, what specific function does it possess and is its function conserved? Animal mtDNA replication is not coupled to cell cycle, yet, the activity of pol y and 89 the expression of the pol 7 genes are clearly developmentally regulated (Wemette and Kaguni, 1986; Ye, et al., 1996). The expression of the pol 7 genes, mitochondrial targeting of precursors of both subunits, and holoenzyme assembly are important steps to produce the functional pol y and very likely regulated in response to the cellular requirement for mitochondrial function. The accuracy and efficiency of each step certainly affects the enzymatic activity of pol y, and thus has impact on genomic stability of mtDNA. DNA pol y represents less than 1% of the total DNA polymerase activity in cells, and the low abundance of pol 7 makes it very difficult to obtain sufficient native enzyme to conduct structure-function studies. The baculovirus expression system provides a eukaryotic environment for recombinant protein production. The advantageous features of this system include high-level expression, capacity for post-translational modification, simultaneous expression of multiple genes, and subcellular compartmentalization of recombinant proteins. Many examples of successful production of active enzymes have been reported, including the expression of human pol 0t (Copeland and Wang, 1991), replication factor C complex , and recently the catalytic subunit of human pol y (Longley, et al., 1998b). However, few mitochondrial proteins have been expressed using this system, and no multi-subunit mitochondrial enzyme has been expressed and purified. We have achieved the baculovirus overexpression, purification and biochemical characterization of the recombinant holoenzyme of Drosophila pol y from insect cells. We also examined holoenzyme reconstitution in both the mitochondria and cytoplasm of infected Sf-9 insect cells. 90 EXPERIMENTAL PROCEDURES Materials Enzymes and Proteins - native Drosophila pol 7 Fraction VI was prepared as described by Wernette and Kaguni (Wernette and Kaguni, 1986). Polyclonal antibodies raised against bacterial produced recombinant or or B subunit were prepared and purified by the method of Olson et a1 (Olson, et al., 1995). Nucleotides and Nucleic Acids - Baculovirus transfer vector pVL1392 / 1393, and linearized wild type baculovirus AcMNPV DNA (BaculoGold) were purchased from PharMingen. Wild type baculovirus AcMNPV was generous gift from of Dr. Suzanne Thiem, Department of Entomology, Michigan State University. Synthetic oligodeoxynucleotides as indicated below were synthesized in an Applied Biosystems Model 477 oligonucleotide synthesizer. Insect Cells and Tissue Culture Medium - Sf—9 ( Spodopterafrugiperda ) cells were kind gift of Dr. Suzanne Thiem, Department of Entomology, Michigan State University. TC-100 insect cell culture medium and Fetal bovine serum were from GIBCO-BRL. Insect cell transfection buffer and Grace's medium were from PharMingen. Chemicals - SeaKem ME low melting agarose was purchased from FMC. Amphotericine, Penicillin—G, Streptomycin, tryptose broth were from Sigma. Protease inhibitors PMSF, sodium metabisulfite, and leupeptin were purchased from Sigma, J. T. Baker Chemical Co., and Peptide Institute, Minoh-Shi, Japan respectively. 91 Methods Construction of Recombinant Baculoviruses - Transfer plasmid vectors containing either the complete coding sequence or coding sequence with N-terminal deletion of the mitochondrial import presequence of both subunit of pol 7 were prepared by standard DNA manipulation. An Nde I fragment containing the complete coding sequence of the or subunit with or without N-terminal deletion was released from E. coli expression pET plasmid vector DNA (Lewis, et al., 1996) and subsequently was subcloned into the EcoR I site of the transfer vector pVL1393 by blunt end ligation after both the vector and insert DNA were repaired with Klenow to generate pVL9301 and pVL930t-NL. A restriction fragment of Xba I and BamH I from E. coli expression pET plasmid vector containing either the complete coding sequences or coding sequence with N-terrninal deletion of mitochondrial import presequence of the B subunit was directionally cloned into the Xba I and BamH I sites of the transfer vector pVL1392 to obtain pVL92B and pVL92 B-NL. The transfer plasmid containing an N-terminal His-tag and deletion of mitochondrial presequence of the (1 subunit (pVL930t-NLNHis) was obtained by replacing the Xba I fragment of pVL930t (representing the N-terminal portion of the coding sequence) with an Xba I fragment from an E. coli expression vector (pET28a-0tNL). The transfer plasmid containing C-terminal His-tagged or subunit (pVL930t-CHis) was prepared by PCR- mediated site-directed mutagenesis to insert the His-tag coding sequence to the C-terminus. Partial DNA sequence of the plasmid constructs were obtained to confirm the correct 92 Fig. 1 Baculovirus transfer vectors. The complete coding sequences of or and B subunits of Drosophila pol 7 were subcloned from their respective bacterial expression plasmids into the transfer vector pVL1392/ 1393 to obtain pVL930t and pVL92B. The mitochondrial presequences of both subunits were deleted from their N-termini and a translation initiation codon was added to obtain pVL930t-NL and pVL92B-NL respectively. A (His) 6 coding sequence was inserted at the N-terminus of pVL930t-NL sequence or C- terrninus of pVL930t sequence to obtain pVL93or-NLNHis and pVL930t-CHis. 93 Baculovirus transfer VCCIOI‘ S Catalytic subunit constmcts M a I i M (lotus I E? M (His)6 (XNL l I M aNlNHis 1521 I (H is)6 Accessory subunit constructs M l3 I l M BNL [ 1 FIGURE 1 94 orientation of the insertion and the preservation of correct reading frame and translational initiation and stop codons. Linearized wild type baculovirus DNA (BaculoGold, 0.5 ug, from PharMingen) and purified transfer plasmid DNA containing different constructs encoding a or B subunit (2 ug) were co-transfected in transfection buffer ( 25mM Hepes pH 7.1, 125mM CaCl 2, 140mM NaCl ) at 27 0C for 4 hours following manufacture’s recommendation. The putative recombinant viruses were plaque purified. The resulting recombinant viruses were designated as AcNPVor, AcNPVB, AcNPVa-NL, AcNPVB-NL, AcNPVOt-CHis, AcNPVor-NLNHis for viruses encoding full length at subunit (p125), full length B subunit (p35), a subunit without mitochondrial import presequence (p125NL), B subunit without mitochondrial import presequence (p125NL), at subunit with C-terminal His tag (p125CHis), 0t subunit with N-terminal His tag and presequence deletion respectively (p125NLNHis, see Figure 1). All viruses were amplified in Sf-9 cells to titers of 5x107 to 1x108 plaque forming units/ml. Cell Culture and Production of Recombinant a and B subunit- Sf-9 (Spodoptera frugiperda ) cells are grown in TC-lOO insect cell culture medium containing 10% fetal bovine serum at 27°C. Sf-9 cells were infected with recombinant virus with the multiplicity of infection (MOI) of 5 in TC-lOO medium supplemented with 10% fetal bovine serum. Cells ( about 1 X10° cells/ml ) were collected by centrifugation at low speed (1x1000g) and 95 lysed in 1X SDS Laemmli gel loading buffer or washed with PBS buffer and frozen in liquid nitrogen and stored at -80° C. The overproduction of recombinant form of at and B subunit was analyzed by immunoblotting with subunit-specific antibodies. Mitochondrial Extraction - Cells collected from 50 ml culture were homogenized in HB buffer ( 15 mM HEPES pH 8.0, 2 mM CaClz, 5 mM KCl, 0.5 mM EDTA, 280 mM Sucrose, 1 mM PMSF, 0.5 mM DTT, 10 mM Na bisulfite, 2 jig/ml leupeptin) or EB buffer (SOmM Tris-HCl pH 7.5, 50 mM KCl, 280 mM Sucrose, 1 mM PMSF, 5 mM B- mercaptoethanol, 10 mM Na bisulfite, 2 rig/ml leupeptin). Mitochondria were pelleted at 7,800xg after removing nuclei and unbroken cells. Mitochondrial fraction was extracted with 2% Na cholate in extraction buffer ( 25 mM HEPES pH 8.0, 10% glycerol, 2 mM EDTA, 300 mM NaCl, 0.5 mM DTT, 1 mM PMSF, 10 mM Na bisulfite, and 2ug/ml leupeptin ). DTI‘ was replaced by 5 mM B-mercaptoethanol and EDTA was omitted in Ni- NTA agarose affinity purification protocol used for His-tagged protein. Both cytoplasmic and mitochondrial fractions were centrifuged at 100,000 xg to remove insoluble materials. Purification of Recombinant Drosophila pol yfrom the cytoplasm of Sf-9 cells - All purification operations were all performed at 0-4 °C. Sf-9 cells infected with recombinant baculovirus encoding the catalytic and accessory subunit were harvested 48 hours post infection. The cells were pelleted and washed with equal volume of cold PBS buffer. The cell pellet (1x109 cells) was resuspended in 20 ml of extraction buffer (50 mM Tris-HCl, pH 7.5, 100 mM KCl, 280 mM Sucrose, 5 mM EDTA, 2 mM DTT, 10 mM Na metabisulfite, 1 mM PMSF, and 2ug/ml leupeptin) and lysed by 20 strokes in a Dounce 96 homogenizer. The homogenate was centrifuged at 3000 rpm, 3 0C, in a SS-34 rotor for 7 min. The pellet was resuspended in 14 ml of extraction buffer and re-homogenized and centrifuged as above. The combined supernatant (Fraction Ial , 34 ml) was centrifuged at 10,000 rpm, 3 °C, for 15 min to pellet the mitochondria. The supernatant (Fraction Ia2) was centrifuged in a Beckman 60Ti rotor at 100,000 X g to obtain cytoplasmic soluble fraction (Fraction Ia3, 2.1 mg/ml of protein) and the mitochondria pellet was washed twice with 50 ml of extraction buffer and frozen in liquid nitrogen and stored at -80 °C. Purification of recombinant pol y holoenzyme was conducted as described (Wernette and Kaguni, 1986) with the following modifications. Soluble (S100) cytoplasmic fraction (Fraction Ia3) was adjusted to an ionic equivalent of 100 mM potassium phosphate and loaded onto a phosphocellulose column (15 ml, 6 mg of protein per packed ml of phosphocellulose resin) equilibrated with 80 mM potassium phosphate buffer at a flow rate of 12 ml/h. The column was washed with 45 ml of 100 mM potassium phosphate buffer at a flow rate of 30 ml/h. Proteins were eluted with 44-ml linear gradient from 150 to 350 mM potassium phosphate buffer at a flow rate of 30 ml/h and active fractions (#17 - 28) were pooled (Fraction II, 20 ml) and adjusted with 80% sucrose to a final concentration of 10%. After addition of 1.2 volumes of saturated ammonium sulfate, pH 7.5, and incubation on ice for 2 h, the precipitate was collected by centrifugation at 96,000 X g for 30 min at 3 °C. The protein was resuspended in 2.0 ml of 10 mM K phosphate buffer containing 45% glycerol, and stored at -20 °C (Fraction IIb). To achieve better precipitation, BSA (0.5 mg/ml) was added to the supernatant and the above procedure was repeated to obtain 4 ml of Fraction IIb (0.46 mg/ml). The Fraction IIb was dialyzed in 250 m1 dialysis buffer (10 mM K phosphate, pH 7.6, 20% glycerol, 2 97 mM EDTA) in a collodion bag (Mr cutoff, 25,000 kDa) until an ionic equivalent of 85 mM KCl was reached. It was then loaded onto a single-stranded DNA-cellulose column (1.8 ml, 1mg of protein per packed ml of resin) equilibrated with 20 mM K phosphate buffer at a flow rate of 1.3 ml/h. The column was washed with 4 ml 20 mM K phosphate buffer containing 100 mM KCl at 2.7 ml/h followed by successive elution with 20 mM K phosphate buffer containing 250 mM KCl (8 ml at 4ml/h), 600 mM KCl (6 ml at 4ml/h), and 1 M KCl (4 ml at 5m1/h). Active fractions were pooled as Fraction III (# 17 - 27, 5 ml). Solid ammonium sulfate was added to Fraction III from the ssDNA-cellulose column to final concentration of 0.4 mg/ml. After stirring for 20 min on ice, the suspensions were centrifuged at 20,000 rpm, 3 °C, for 10 min in a Beckman Ti60 rotor. The supernatant was loaded onto an Octyl-Sepharose column (0.5ml) which was equilibrated with 20 mM K phosphate buffer at a flow rate of 0.5 ml/h. The Octyl-Sepharose column was washed with 2 ml of equilibration buffer at 2 ml/h and then eluted at the same flow rate with 1.75 ml of buffer containing 0.3% Triton X-100, 2 ml of buffer containing 1% Triton X-100, and 1.5 ml of buffer containing 2% Triton X-100. Fraction IV was then loaded on 12- 30% glycerol gradient and the peak fractions (Fraction V) containing recombinant pol 7 were collected, assayed, pooled, stabilized, and stored at -20 °C or frozen with liquid nitrogen and stored at -80 °C. Ni-NTA agarose affinity purification of recombinant pol ’y - Soluble (8100) cytoplasmic fraction (10 ml) of Sf-9 cells co-infected with viruses was mixed with 50 [.11 of pre-charged Ni-NTA (nitrilo-tri-acetic acid) agarose resin and incubated on ice with gentle shaking for 4 hours. The resin was washed 2 times with 10 ml of buffer (50 mM Tris-HCl pH 7.5,200 mM KCl, 10% glycerol, 1 mM PMSF, 5 mM B-mercaptoethanol, 10 mM Na 98 bisulfite, 2 jig/ml leupeptin, and 5 mM imidazole). Proteins retained on the resin were eluted first with 1 ml of washing buffer (with 50 mM imidazole) followed by second elution with 0.3 ml washing buffer (with 300 mM imidazole). All the washing and elution steps were done on ice and for 30 minutes with gentle shaking. Mitochondria from 150 ml of Sf—9 cells infected with recombinant virus expressing 0t and B subunits were extracted in 4 ml of EB-mito extraction buffer (25mM HEPES pH 8.0, 10% glycerol, 300mM NaCl, 1 mM PMSF, 10 mM Na bisulfite, 2 jig/ml leupeptin). Recombinant pol ywas purified with 100 1.11 of pre-charged Ni-NTA agarose resin following the same procedure as for the cytoplasmic extract except the washing and elution buffer was changed to EB-mito buffer. DNA polymerase assay - DNA polymerase activities were assayed on DNase I- activated calf thymus DNA or singly-primed M13 DNA at 30 - 200 mM KCl as described by Olson (Olson, et al., 1995). The reaction mixtures (0.05 ml) contained 50 mM Tris- HCl pH 8.5, 4 mM MgC12, 10 mM DTT, 400 ug/ml bovine serum albumin, and 20 or 30 BM each of dGTP, dATP, dCTP, and [3H]d'ITP(1000 cpm/pmol). Specific modifications are described in the figure legends. One unit of DNA polymerase activity is defined as the amount that catalyzes the incorporation of 1 nmol of deoxyribonucleoside triphosphate into acid insoluble material in 60 min at 30 °C using DNase I-activated calf thymus DNA as the substrate. 3 ’ -5 ’ exonuclease assay - Reaction mixtures (0.05 ml) containing 50 mM Tris- HCl pH 8.5, 4 mM MgC12, 10 mM DTT, 400 ug/ml bovine serum albumin, and KC] as indicated, 4 11M 5’-end labeled singly-primed M13 DNA containing a 3’-terminal mispair, 99 and 0.1 unit of Fraction VI recombinant pol 7 were incubated for 30 min at 30 °C in the presence or absence of saturating amount of recombinant mtSSB protein. Samples were then made 1% in SDS and 10 mM in EDTA, heated for 10 min at 65 °C and precipitated with ethanol in the presence of 1 ug sonicated salmon sperm DNA as carrier. The ethanol precipitates were resuspended in 80% formamide and 90 mM Tris-borate. Aliquots were denatured for 2 min at 100 °C, chilled on ice and electrophoresed in an 18% polyacrylamide gel (13x13x0.075 cm) containing 7 M urea in 90 mM Tris-borate pH 8.3 and 2 mM EDTA. After electrophoresis, the gel was washed in 15% glycerol for 20 min and exposed at - 80 °C to Kodak X-Omat AR x-ray film using a DuPont NEN Quanta III intensifying screen. Other Methods - Protein concentration was determined by the method of Bradford with Bovine serum albumin as the protein standard. Gel electrophoresis and protein transfer and immunoblotting were performed as described by D. L. Lewis et al. (Lewis, et al., 1996). 100 RESULTS Construction of recombinant baculoviruses and overexpression of recombinant subunits of Drosophila pol y in cultured insect cells Co-transfection of linear baculovirus DNA and transfer vector containing a-subunit or B-subunit coding sequence with or without their mitochondrial presequence (leaders) generated recombinant baculoviruses very efficiently (Figure 1). All recombinant viruses were purified by isolating a single viral plaque and amplified in Sf-9 cells to high titer ( 1X 10 3 pfu/ml). For each recombinant virus, ten to twelve independently isolated clones were amplified and tested for expression of recombinant protein. All of these produced recombinant at or B subunit polypeptides at similar levels (data not shown). Analysis of whole cell extracts of infected Sf9 cells showed that the expression levels of recombinant 0t- or B-subunit polypeptides are similar to that of viral polyhedrin protein. Figure 2 shows an SDS-polyacrylamide gel analysis of whole cell extracts from Sf-9 cells, cells infected with wild-type baculovirus, and cells infected with recombinant baculoviruses encoding at or B subunits in different forms. Cells infected with AcNPVa, AcNPVOt-NL, AcNPVa- CHis, AcNPVa-NLNHis all produced a protein of 125 kDa that was immunoblotted by at subunit-specific antibody. Similarly, cells infected with AcNPVB or AcNPVB-NL produced a protein of 35 kDa that reacted with B subunit-specific antibody. Extracts from uninfected Sf-9 cells and cells infected with wild type virus did not contain any protein that cross-reacted with either or or B subunit-specific antibody. Production of total recombinant 101 Fig. 2 Overexpression of recombinant 0t and B subunits in Sf-9 cells. Sf-9 cells were infected with recombinant baculovirus expressing at or B subunit as full-length protein or with modifications as indicated at the MOI of 5, and harvested 48 hours after infection and total cell extract from 1X105 cells (for immunoblotting in panel B) or 2X10S cells (for staining in panel A) was resolved on 10% SDS-PAGE gels. Lane I represents a control. Lane 2 and 3 represent cell extracts from uninfected cells (C) and cells infected with wild type baculovirus (W). Lanes 4, 5, 6, and7 represent cells expressing 0t subunit as full length (or), C-terminal His-tag fusion (aCHis), N-terminal presequence deletion (OtNL), and N-terminal presequence deletion with His-tag fusion (aNHis). Lanes 8 and9 represent cells expressing B subunit as full length (B) and N-terminal presequence deletion (BNL). 102 Overexpression of recombinant or. and B subunits in Sf-9 cells. 12 3 45 6 7 89 WaaCHisaNLaNI-IisBBNL N. B.Immunoblot 1 2 3 4 5 6 7 8 9 W a. aCHisaNLaNHisB BNL iii» FIGURE 2 103 protein was calculated to be about 2 jig/m1 of cultured cells. Protease degradation of recombinant proteins was observed, and subcellular fractionation experiments indicated that only about 10 to 20 % of recombinant protein was recovered in the soluble fraction. Overexpression in other insect cell lines (Sf21 or High Five) did not improve the solubility or eliminate the proteolysis of recombinant proteins (data not shown). Experiments performed by varying MOI (multiplicity of infection) or adding the protease inhibitor phenylmethylsulfonyl fluoride (PMSF) at lmM into the culture medium also failed to prevent proteolysis (data not shown). Mitochondrial import of recombinant a and B subunits Mitochondria were prepared as indicated in Methods and the crude soluble S-100 extracts were analyzed by immunoblotting. Recombinant a and B subunit are clearly detected in the mitochondrial fraction (Figure 3). Full-length recombinant at or B subunit are competent for mitochondrial import; however, the targeting and import efficiency appears to be suboptimal. Insect cell mitochondria isolated 48 hours post infection contain mostly full length recombinant proteins with minimal levels of degradation (Figure 3), indicating that the mature recombinant a and B polypeptides are protected from protease(s) once they are imported into the mitochondrial matrix. The mitochondrial presequence is necessary for proper mitochondrial import. No recombinant protein is detected in mitochondrial extracts when they are expressed without a mitochondrial presequence at their N-termini (p125NL or p35NL); instead, the recombinant or and Bpolypeptides remained in the insect cell cytoplasm (Figure 3). The estimated yield of recombinant 0t and 104 Fig.3 Mitochondrial import of recombinant or and B subunits. Sf-9 cells were infected with recombinant baculovirus expressing or and/or B subunits as full-length proteins (or or B) or lacking presequences (GLNL or BNL) at the MOI of 5, and harvested 48 hours after infection. Soluble mitochondrial (Mi) and cytoplasmic (Cy) fractions were extracted from 3X107 cells and aliquots of these fractions were resolved on 10% SDS- PAGE gels. The recombinant polypeptides in each fraction were detected by immunoblotting using subunit-specific antibodies. Lane I and 2 represent samples from co-expression of full-length or and B subunits. Lane 3 and 4 represent samples from co- expression of (1 and B subunits lacking presequences. Lane 5, 6, 7, and 8 represent samples from at and B subunits expressed individually and lacking presequences. 105 Mitochondrial import of recombinant or and B subunits FIGURE 3 106 B polypeptides in the mitochondrial fraction is only about 40 ng per ml of cells collected 48 hours post infection, representing ~ 2% of total recombinant protein. Co-expression of recombinant 0t and B subunits reconstitutes pol y holoenzyme Whether the recombinant or and B subunits are properly folded and recombinant heterodimeric pol y holoenzyme is formed in the mitochondria or cytoplasm was determined by glycerol gradient sedimentation. The crude mitochondrial soluble fraction derived from cells infected with both recombinant viruses AcNPVor and AcNPVB was sedimented in a 12-30% glycerol gradient. The recombinant 01 and B subunits were found to co-sediment (Figure 4). Native pol y purified from Drosophila embryos sedimented at the same glycerol density. The identity of recombinant B subunit in the peak fractions was confirmed by immunoblotting using B subunit-specific antibody (data not shown). Identical results were obtained whether the cells were harvested 36, 42, or 48 hours after co-infection (data not shown). Remarkably, recombinant full length or and B subunits are also assembled into heterodimer in the cytoplasm of Sf-9 cells, and the addition of a (His) 6 tag at the C-terminus of the at subunit does not disrupt the protein-protein interaction (data not shown). Recombinant catalytic subunit expressed individually sedimented at lower glycerol density. However, recombinant B subunit expressed individually was largely insoluble and failed to sediment as a discrete species (data not shown). 107 Fig. 4 Reconstitution of recombinant Drosophila pol y heterodimer in mitochondria of Sf-9 cells. Soluble mitochondrial extract from Sf-9 cells infected with recombinant baculoviruses expressing or and B subunits was sedimented in a 12-30% glycerol gradient. Proteins were resolved on 10% SDS-PAGE gels and detected by immunoblotting using subunit-specific antibodies. Numbers labeling each lane represent the fraction numbers. 108 Reconstitution of a recombinant Drosophila pol y heterodimer in mitochondria of Sf-9 cells Glycerol Gradient Sedimentation Analysis Fr# 10 12 14 16 18 20 22 24 26 28 30 a_ -—n l J " ‘ ¢ —— gun-- as:- B- 5‘ . III-r FIGURE 4 109 Interestingly, co-expression of recombinant 0t and B subunits as mature forms lacking their mitochondrial presequences (p125NL/p35NL) does not yield a pol y heterodimer in the soluble cytoplasmic fraction (Figure 5). In fact, removal of the mitochondrial presequence of the (1 subunit alone (p125NL) in co-expression experiments is sufficient to disrupt holoenzyme assembly in the cytoplasm. Soluble 0t subunit without the leader sequence (p125NL) can be purified by chromatography on a phosphocellulose column (Figure 5), although the B subunit alone can not be recovered. Purification of recombinant Drosophila pol 7 Near homogeneous recombinant Drosophila pol 'y holoenzyme was purified directly from the soluble cytoplasmic extract of Sf-9 cells infected with baculoviruses encoding the complete coding sequences of the at and B subunits by sequential chromatography on phosphocellulose, ssDNA-cellulose, and octyl-Sepharose followed by glycerol gradient sedimentation as described in Methods. Table 1 and Figure 6 summarize the results of one purification. The two subunits of pol yremained tightly associated in the same apparent stoichiometry throughout the purification. The purified recombinant holoenzyme is an active DNA polymerase with proofreading 3’ - 5’ exonuclease activity. Furthermore, the specific activity of the recombinant enzyme is 20,000 units/mg, which is similar to that of native pol y. The heterodimeric form of recombinant pol 'y with the catalytic subunit expressed as a C-terminal his-tag fusion protein can also be purified as an active enzyme using N i-NTA agarose affinity chromatography (data not shown), although 110 Fig. 5 Co-expression of or and B subunits lacking mitochondrial presequences. Soluble cytoplasmic fraction from 3X107 Sf-9 cells infected with recombinant baculoviruses expressing 0t and B subunits was chromatographed on a phosphocellulose column. The fractions collected from the column were resolved on 10% SDS-PAGE gels and the at and B subunits were detected by immunoblotting. Mi and Cy represent mitochondrial and cytoplasmic soluble fractions. F and W represent flowthough and wash fractions. Numbers above each lane represent the eluted fraction number. In and M represent the cytoplasmic insoluble fraction and a molecular weight control respectively. 111 Co-expression of or and B subunits lacking mitochondrial presequnces MiCyFWl3579111315171921WInM n. . ‘ . , . . . ' v . FIGURE 5 112 Table I Purification of recombinant Drosophila pol y from baculovirus-infected Sf-9 cells Fraction Volume Protein Activity Specific Yield Activity (ml) (mg) (units) (units/mg) (%) I. Soluble cyto. extracta 34 71.4 8092 1 13 100 H. Phosphocellulose and 4.5 1.84 974 529 12 ammonium sulfate III. ssDNA-cellulose l 1.2 0.69 748 1084 9 IV. Octyl-sepharose 3.0 ND 159 ND 2 V. Glycerol gradient 9.0 0.0075 149 20,000 2 aFraction I was prepared from 0.5 L of infected Sf-9 cells 113 Fig. 6 Purification of recombinant Drosophila pol 7. Fraction VI of native (lane 1, 10 units) and fraction V of recombinant (lane 2, 9 units ) pol 7 were electrophoresed in a 10% SDS-PAGE gel and the proteins were stained with silver. Fraction V of recombinant pol 7 (lane 3, 1.5 units) was also probed with subunit-specific antibodies. 114 Purification of recombinant Drosophila pol y FIGURE 6 115 the specific activity is ~10-fold lower. Stimulation of both DNA polymerase and 3’-5’ exonuclease activities of recombinant Drosophila pol y by salt and by mtSSB The recombinant pol y holoenzyme exhibits very similar biochemical properties to that of native pol y purified from Drosophila embryos. Its DNA polymerase activity is stimulated 8 fold by elevated salt (200 mM KCl) on DNase I-activated calf thymus DNA and 30 fold by mtSSB on singly-primed M13 DNA. Its mispair-specific 3’ - 5’ exonuclease activity is also stimulated by salt and mtSSB (data not shown). 116 DISCUSSION An efficient and reliable system for overexpression of recombinant proteins, particularly those proteins with very low abundance, will provide the foundation for further structural and functional studies. We have pursued overexpression of recombinant Drosophila pol 7 since we isolated the cDNAs for both subunits. The baculovirus expression system can produce both subunits of pol 'y in soluble form and yield an active recombinant heterodimeric enzyme. (At the same time, we failed to achieve in vivo reconstitution of heterodimeric holoenzyme by bacterial expression (Wang, et al., 1997b). The recombinant subunits of pol y are targeted and imported into mitochondria of Sf—9 cells whether they are expressed individually or simultaneously. The successful reconstitution of holoenzyme in the mitochondria of Sf-9 cells suggests that the cellular machinery for mitochondrial localization and processing is conserved between Drosophila and Spodoptera frugiperda. The low yield of recombinant pol y in Sf—9 cell mitochondria is probably the result of the lytic nature of baculovirus infection. It is recognized that host protein synthesis is greatly reduced 24 hours after infection when viral late genes dominate mRNA transcription. The fact that the recombinant holoenzyme can be purified from the soluble cytoplasmic fraction and exhibits nearly identical chromatographic behavior and biochemical and physical properties suggests strongly that the recombinant holoenzyme is able to achieve proper folding in the cultured insect cells, and demonstrates further that the B subunit is an essential component of pol y. The successful in vivo reconstitution of holoenzyme provides us with a useful system to study mutant forms of holoenzyme and to examine subunit interactions in pol y. 117 We have constructed recombinant baculoviruses containing the corresponding coding sequences of the mature form of both subunits in an effort to produce and purify the recombinant enzyme without the N-terminal mitochondrial presequences, to compare the recombinant and native pol 7, because the latter purified from embryonic mitochondria lacks the presequences. However, when both subunits were co-expressed without their presequences, the solubility of both recombinant proteins and holoenzyme reconstitution was greatly reduced. The deletion of the presequence in the catalytic subunit alone is sufficient to disrupt the proper association of two subunits in the cytoplasm. The exact role of the presequence in promoting holoenzyme assembly is not clear. However, it is possible that the targeting of nascent recombinant protein to the mitochondria itself brings the two subunits in close proximity, thus aiding heterodimer formation. The low efficiency of mitochondrial import of recombinant pol y and the discovery that the heterodimeric form of recombinant pol y is reconstituted in the cytoplasm of insect cells prompted us to purify recombinant pol 7 directly from the soluble cytoplasmic fraction. The recombinant pol y purified in this study is thus slightly different structurally from native pol y, as it contains the mitochondrial presequences in both subunits. The presequence in the catalytic subunit represents only 9 amino acid residues, and the predicted presequence in the accessory subunit is 11 amino acid residues. One modified human recombinant catalytic subunit expressed and purified from insect cells is an N-terminal his-tag fusion protein; there the addition of the his-tag does not change its enzymatic properties (Longley, et al., 1998b). We did not detect a significant alternation of the catalytic properties of our recombinant pol y as compared with the native enzyme. In fact, the addition of a his-tag at the C-terminus of the catalytic subunit does not disrupt the subunit association. The lower than expected 118 specific activity of this form of enzyme was likely the result of its purification by Ni-NTA affinity chromatography. The human pol y catalytic subunit has been purified as recombinant enzyme from Sf-9 cells and from cultured Hela cells. They exhibit similar enzymatic properties. However, its DNA polymerase activity is salt sensitive (Longley, et al., 1998b) and it is not known whether mtSSB can stimulate its DNA polymerase and 3’ - 5’ exonuclease activities. Our initial characterization of recombinant Drosophila pol 7 clearly shows that it exhibits very similar biochemical properties relative to native pol y in terms of its response to stimulation by both elevated salt and mtSSB. Whether the difference observed in our study and the others reflects the functional significance of the B subunit remains to be determined. However, these results suggest the possibility that physical interaction of two subunits is important in modulation of the catalytic properties of the catalytic subunit. In summary, we have established an effective eukaryotic expression system to produce recombinant pol y and study holoenzyme assembly. To our knowledge, this work represents the first example of reconstitution of a multi-subunit mitochondrial enzyme using the baculovirus expression system. 119 FUTURE RESEARCH We are currently performing overexpression and purification of the catalytic subunit alone to compare the biochemical properties of the catalytic subunit versus holoenzyme. Whether the B subunit plays a role in modulating overall activity, and/or fidelity and processivity of DNA synthesis will be tested when purified catalytic subunit becomes available. Mutant holoenzyme lacking 3’-5’ exonuclease activity will also be produced and its enzymatic properties will be examined. Structure-function studies with recombinant enzymes will be carried out following the experimental approaches described in Chapter II. 120 LIST OF PUBLICATIONS--YUXUN WANG Olson, M.W., Wang, Y., Elder, RH, and Kaguni, LS. (1995) Subunit structure of mitochondrial DNA polymerase from Drosophila embryos: Physical and immunological studies. J. 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