m” ‘2‘». , w a {5 néfiwmgzfizfifigfigm “min; ‘ ‘3 - 7‘ .w. “’ fail- . it“ '1 \L _’v.‘{“,~fi.-x.< ‘ 'V iaflgus‘dw “rawi‘ggnj. {“é‘wfi‘wfi Jiakiu. 45,-“ ~.... ~ "3 . ~‘QL J‘ a» “$354: ‘ ' .JJ \ 333:9 ,- "‘“ 43$. . ‘ “aim“. v, ‘..m 1 :ng; 2:1»; V559. agar: » r- . ‘ “3:: win _‘:‘ -. 'z: _ .4. ‘63. w—vcfiflfi', , ‘6‘ n} ‘~f‘+‘°§‘.z:."’."" ' “in: '3‘: it t. - Ti . 6 fig???“ I .‘vré ,. by "m ‘; 4V." ‘4 .- J‘s). ,’ .. ”a, . 3 H"? . n _ gm, . ~ 44:1?- 7i?¥?~%»‘s%”'5 . I.¢.;::.K'.-;1‘..J.‘.‘.‘ "@134“ ‘S” , \. fig? 2 éé‘ 1'- - ‘7 Qfifi‘s’i‘ 7.; "eves? on w... .~ Illlfll’illlllllllllililll'lllllllllllillll 293 01015 2134 This is to certify that the dissertation entitled Processivity of Mitochondrial DNA Polymerase From Drosophila Embryos: Effects of Reaction Conditions, DNA-Binding Proteins and Enzyme Purity presented by Andrea Jean VonTom has been accepted towards fulfillment of the requirements for Ph . D . degree in Biochemistry We .5- baa-r". Major professor Date 5/ 5:144- MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 LIBRARY - Michigan State University PLACE ll RETURN BOXto mummi- Mountain your record. TO AVOID FINES mm on or baton dd. duo. DATE DUE DATE DUE DATE DUE MSU In An Affirmative MONEquul Oppommly lm WIN-94 PROCESSIVITY OF MITOCHONDRIAL DNA POLYMERASE FROM DROSOPHILA EMBRYOS: EFFECTS OF REACTION CONDITIONS, DNA-BINDING PROTEINS AND ENZYME PURITY By Andrea Jean VonTom A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1994 ABSTRACT PROCESSIVITY OF MITOCHONDRIAL DNA POLYMERASE FROM DROSOPHILA EMBRYOS: EFFECTS OF REACTION CONDITIONS, DNA-BINDING PROTEINS, AND ENZYME PURITY By Andrea Jean VonTom Mitochondrial DNA polymerase from Drosophila melanogaster embryos was characterized with regard to its mechanism of DNA synthesis under the influence of a variety of compounds in moderate salt, where the enzyme is most active and only moderately processive, and in low salt, where it is less active yet most processive. Disparate activity and processivity optima were obtained in low salt in the presence of varying pH or MgC12 and ATP concentrations; in moderate salt, optimal activity and processivity were achieved coincidentally. Whereas no correlation between processivity and activity optima was observed upon addition of polyethylene glycol, the optima were coincident at both salt levels upon addition of glycerol. None of the reaction conditions examined allowed DNA polymerase ‘y to exhibit maximal activity and processivity concurrently; maximal activity was always achieved in moderate salt and highest processivity in low salt. 1 Addition of sin gle-stranded DNA binding protein from Escherichia coli resulted in the concurrent achievement of maximal activity and processivity by pol yin low salt, suggesting that 7 polymerase, like other DNA polymerases, associates with accessory factors in vivo to catalyze efficient and processive DNA synthesis. The processivity of and intrinsic rate of nucleotide polymerization by mitochondrial DNA polymerase were increased only 3-fold by single-stranded DNA binding protein, while the rate of initiation of DNA synthesis was increased 25-fold. Maximal stimulation of 'y polymerase was achieved following a lag time that correlated with the time required for DNA polymerase to productively bind DNA. The high degree of amino acid sequence similarity between mitochondrial and Escherichia coli single-stranded DNA binding proteins suggests that mitochondrial sin gle- stranded DNA binding protein may affect pol ‘y similarly. The ability of mitochondrial DNA polymerase to copy singly-primed DNA was enhanced then diminished during the course of purification, suggesting the loss of an accessory factor. While stimulatory and DNA binding activities co-sedimented with a 145 kDa polypeptide, gel filtration chromatography resulted in their separation. While a potent endonuclease activity was responsible for DNA polymerase stimulatory activity, purification of the 145 kDa polypeptide by immunoblot confirmed its identity as an apparent template-primer specific DNA binding protein. For My Students iv ACKNOWLEDGMENTS I would like to thank Laurie Kaguni for her friendship, guidance, and perseverance over the last five years. If not for her persuasive arguments, I would have left graduate school years ago unable to fulfill my only wish upon enrolling: to teach college science. Laurie has had a huge influence on my perception of life and, at the risk of sounding maudlin, I will always remember the lessons that I have learned from her. Thanks to Jon Kaguni and members of both Kaguni labs (Matthew Olson, Dave Lewis, Carol Farr, Rhod Elder, Angie Kollhoff, J.J. Wang, Kevin Carr, Mark Sutton, Cindy Petersen, Carla Margulies, Wenge Zhang, Jarek Marszalek, and Ted Hupp), as well as the members of my committee (Shelagh Ferguson-Miller, Justin McCormick, Alex Raikhel, and John Wilson) for critically analyzing my work and aiding in my development as a scientist. My involvement with the M.S.U. Women in Science and Engineering group has, over the last couple of years, given me a sense of purpose which has helped me muddle through what sometimes seemed purposeless. The support I received from them, and especially from Eileen Van Tassell, is greatly appreciated. Clarence Suelter deserves much recognition for his efforts to make my teaching goals more attainable. His guidance, support, and willingness to offer a course for graduate students interested in teaching college science has been a bright spot in my dark experience that was graduate school. Additionally, I wish to thank my classmates, Cindy Petersen and Marty Regier, who share both my love of teaching and my frustration with grad school, for their willingness to listen anytime I wanted to complain and to talk when I needed to know that I was not alone. My life away from the lab was probably more important in keeping me focused on completing my degree than my life in it. Therefore, I'd like to thank Isaac and Doppler who, even though they have left me in my hour of greatest need, provided me with much needed fur therapy over the last five years, Monica Gloudemans for her friendship and enthusiasm, Diane Cox for her supportive and understanding nature, the "Club Eleven" soccer team (especially Denise Egelund and Michelle Davenport) for providing a creative outlet for my frustration, and Colleen Curry for being a good friend and a good example. Angie Kollhoff has been everything anyone could ever want in a friend over the last year and a half. I don't think I could find words enough to thank her, sol guess I'll just say thanks and let our futures say the rest. My husband, Tim, deserves many many many thanks. Along with Laurie, he managed to pressm'e me into staying in school at the times that I most wanted to leave, even though it meant prolonged separation for us. His desire for what would eventually be good for me outweighed that which. would have been good for him. For this I am truly grateful. Finally, I'd like to express my appreciation to my family for their love and support. I've been gone too long, now it's time to go home. PREFACE Chapter II of this dissertation, along with figure 21 (chapter IV), was reproduced with permission from the Journal of Biological Chemistry. Williams, Andrea J ., Wemette, Catherine M., and Kaguni, Laurie S. (1993) Processivity of Mitochondrial DNA Polymerase from Drosophila Embryos: Effects of Reaction Conditions and Enzyme Purity. J. Biol. Chem. 268, 24855-24862. Chapter III of this dissertation, along with experiments examining the effects of Escherichia coli sin gle-stranded DNA binding protein on the 3'—)5' exonuclease activity of mitochondrial DNA polymerase, will be submitted to the Journal of Biological Chemistry. Williams, Andrea 1., and Kaguni, Laurie S. (1994) Stimulation of Mitochondrial DNA Polymerase from Drosophila Embryos by Single-Stranded DNA Binding Proteins. J. Biol. Chem. TABLE OF CONTENTS List of Figures ........................................................................................ xiv List of Abbreviations ................................................................................ xvi Chapterl Literature Review ................................................................... 1 DNA Replication ........................................................... 2 DNA Synthesis in Escherichia coli ...................................... 6 Replicative DNA polymerase .................................... 6 DNA polymerase accessory factors ............................ 11 DNA Synthesis in Bacteriophage T4 .................................... l4 Replicative DNA polymerase .................................... 14 DNA polymerase accessory factors ............................. 15 DNA Synthesis in Simian Virus 40 ...................................... 18 Multiple DNA polymerases in eukaryotic DNA replication ...................................... 19 Leading strand DNA polymerase ................................ 20 DNA polymerase accessory factors ............................. 21 Lagging strand DNA polymerase ............................... 23 DNA polymerase-primase ....................................... 24 Single—stranded DNA binding protein ......................... 25 Mitochondrial DNA replication .......................................... 27 Mitochondrial RNA polymerase and DNA primase ......... 34 Mitochondrial DNA topoisomerase ............................ 37 Mitochondrial DNA helicase .................................... 38 Replicative DNA polymerase ................................... 38 DNA polymerase accessory factors ............................ 40 Chapter II Processivity of Mitochondrial DNA Polymerase From Drosophila Embryos: Effects of Reaction Conditions ......................... 42 Introduction ................................................................ 43 Experimental Procedures ................................................. 45 Materials ........................................................... 45 Nucleotides and nucleic acids .......................... 45 Enzymes .................................................. 45 Chemicals ................................................. 45 Methods................. ..................... l ...................... 45 DNA polymerase y assay ............................... 45 Analysis of products of processive DNA synthesis by gel electrophoresis ....................... 46 Results ...................................................................... 47 Drosophila DNA polymerase 'y is highly processive at KCl levels suboptimal for DNA synthesis .................................................. 47 pH has little effect on the processivity of Drosophila mitochondrial DNA polymerase .......... 47 DNA synthetic product length is diminished with increasing MgClz concentrations ................ 50 ATP specifically stimulates DNA synthesis . by Drosophila y polymerase ............................ 55 Effects of macromolecular crowding agents .......... 55 Glycerol stimulates DNA polymerase activity and processivity concurrently .......................... 58 Limited template-primer availability does not alter the processivity of Drosophila y polymerase ............................................... 58 Discussion ................................................................. 66 ix Chapter III Escherichia coli Single-stranded DNA Binding Protein Stimulates Mitochondrial DNA Polymerase From Drosophila Embryos ................. 71 Introduction ............................................................... 72 Experimental Procedures ................................................ 75 Materials .......................................................... 75 Nucleotides and nucleic acids ......................... 75 Enzymes ................................................. 75 Methods .......................................................... 75 DNA polymerase 'y assay ............................. 75 Analysis of products of processive DNA synthesis by gel electrophoresis ...................... 76 Time course of pol 7 DNA synthesis ................ 76 Primer extension assay ................................ 76 Results .................................................................... 78 E. coli SSB stimulates DNA polymerase 7 activity ......... 78 SSB stimulation of DNA polymerase 7 activity exhibits a lag time ............................................... 83 SSB has little effect on the processivity of DNA polymerase y .......................................... 83 SSB increases the rate of initiation of DNA synthesis by y polymerase .............................. 88 SSB has little effect on the intrinsic rate of DNA synthesis by DNA polymerase 'y ....................... 88 Drosophila SSB and bacteriophage T4 gp32 stimulate mitochondrial DNA polymerase ................... 94 E. coli DNA polymerase III subunits had no effect on pol 7 activity or DNA binding ...................... 97 Discussion ................................................................ 98 X Chapter IV Identification and Purification of a Template—Primer DNA Binding Protein from Drosophila Mitochondria ........................................ 106 Introduction ............................................................. 107 Experimental Procedures .............................................. 109 Materials ........................................................ 109 Nucleotides and nucleic acids ....................... 109 Enzymes and antiserum .............................. 109 Methods ......................................................... 109 DNA polymerase y assay ............................ 109 Analysis of products of processive DNA synthesis by gel electrophoresis .............. 112 DNA polymerase stimulatory assay ................. 113 Construction of synthetic templates for DNA binding ....................................... 113 DNA binding assay .................................... 114 - Glycerol gradient sedimentation ..................... 114 Gel filtration chromatography ........................ 114 Purification of D. melanogaster DNA polymerase y ..................................... 114 Production of antiserum ............................... 115 Purification of DBP-145 ............................... 116 DBP-l45 peptide sequences ........................... 116 Results ..................................................................... 117 The ability of mitochondrial DNA polymerase to copy efficiently single-stranded DNA is enhanced, then diminished during purification ........................... 117 Mitochondrial DNA polymerase stimulatory activity co-sediments with DNA binding activity ..................... 117 DNA binding and DNA polymerase stimulatory activities are separated by gel filtration chromatography ............... 120 x1 Production of polyclonal antiserum raised against the 145 kDa polypeptide ........................................ 128 Purification of the 145 kDa putative DNA binding protein ............................................ 128 Physiological role of DNA binding protein-145 ............. 134 Discussion ................................................................. 135 Chapter V Screening a cDNA Expression Library for Mitochondrial DNA Polymerase From Drosophila ............................................. 141 Introduction ............................................................... 142 Experimental Procedures ................................................ 144 Materials .......................................................... 144 kgtll cDNA library .................................... 144 Nucleic acids ............................................ 144 Enzymes ................................................. 144 Methods .......................................................... 144 Screening the Agtll cDNA library ................... 144 Antibody affinity purification ......................... 145 Polymerase chain reaction analysis of insert size ............................................. 145 Overproduction and sizing of B—galactosidase- pol 7 fusion protein ..................................... 145 Subcloning, DNA sequencing and alignment with yeast 7 polymerase ................................ 146 Results ..................................................................... 147 Screening a lgtll cDNA library ............................... 147 Verification of putative positive clones ........................ 147 Analysis of ltdm-y7l ............................................ 147 XII Discussion ................................................................. 153 ChapterVI Summary and Perspectives ....................................................... 155 List of References .................................................................................... 160 Chapter I 4. 5 . Chapter II 6. 10. 11. 12. Chapter III 1 3. 14. LIST OF FIGURES Advancing DNA replication fork of E. coli .................................... Looping model for coordinated DNA synthesis of the leading and lagging DNA strands ........................................ Representation of vertebrate mitochondrial DNA ............................. Model of mouse mitochondrial DNA replication .............................. Model of Drosophila mitochondrial DNA replication ......................... KCl alters the rate and processivity of DNA synthesis by Drosophila pol y ............................................. pH affects the rate and processivity of DNA synthesis by Drosophila pol y ............................................. MgClz alters the rate and processivity of DNA synthesis by Drosophila pol y ............................................. Effect of ATP, GTP, and ADP on the rate and ATP on the processivity of DNA synthesis by Drosophila pol y .......................... PEG alters the rate and processivity of DNA synthesis by Drosophila pol y ............................................ Glycerol increases the rate and processivity of DNA synthesis by Drosophila pol y ............................................ Varying the pol yttemplate-primer molecular ratio has little effect on the processivity of DNA synthesis by Drosophila pol y .......... E. coli SSB stimulates the rate of DNA synthesis by Drosophila pol “y .............................................................. E. coli SSB lowers the KCl optimum of Drosophila pol y .................. xiv 8 30 33 36 49 52 54 57 6O 62 65 80 82 15. 16. 17. 18. 19. Chapter IV 20. 21. 22. 23. 24. 25. 26. Chapter V 27. 28. E. coli SSB stimulation of Drosophila pol y exhibits a lag time ............ 85 E. coli SSB increases the processivity of DNA synthesis by Drosophila pol y ............................................ 87 E. coli SSB increases the initiation of DNA synthesis by Drosophila pol 'y ............................................ 90 Effect of E. coli SSB on a time course of DNA synthesis by Drosophila pol y ............................................ 93 Drosophila SSB and bacteriophage T4 gp32 stimulate Drosophila pol y ....................................................... 96 Synthetic substrates for DNA binding ......................................... 111 Effect of enzyme purity on the efficiency and processivity of DNA synthesis by Drosophila pol 7 on singly-primed M13 DNA .......... 119 Co-sedimentation of DNA polymerase stimulatory and DNA binding activities ...................................................... 122 Gel filtration of DNA polymerase stimulatory and DNA binding activities ...................................................... 124 Specificity of DNA binding by pol 7 and the 145 kDa DNA binding activity .............................................. 127 Reactivity and specificity of polyclonal antiserum raised against the 145 kDa polypeptide ......................................... 130 Co-chromatography of DNA binding activity - and 145 kDa polypeptide ......................................................... 133 Reactivity of fusion protein affinity-purified antibodies with D. melanogaster DNA polymerase y ...................................... 149 Amino acid sequence alignment of Adm-Y7 1 with yeast mitochondrial DNA polymerase catalytic subunit ........................ 151 XV apu ATP A'I'Pase CAPS dATP DBP-145 dCI'P dGTP D. melanogaster DNA DNase I dNMP dNTP DSSB d’ITP E. coli HSV-l mtDNA mtS SB NAD nt LIST OF ABBREVIATIONS angstrom average processive unit adenosine triphosphate adenosine triphosphatase 3-(cyclohexylamino) propanesulfonic acid deoxyadenosine triphosphate 145 kilodalton DNA-binding protein deoxycytosine triphosphate deoxyguanosine triphosphate Drosophila melanogaster deoxyribonucleic acid deoxyribonuclease I deoxynucleoside monophosphate deoxynucleoside triphosphate Drosophila single-stranded DNA binding protein deoxythymidine triphosphate Escherichia coli gene product herpes simplex virus-1 kilodalton mitochondrial DNA mitochondrial SSB nicotinamide adenine dinucleotide nucleotide xvi PCNA PEG pfu pol a pol Y pol 5 pol e pol III RF-A RF-C S. cerevisiae SSB ssDNA SV40 topo 11 X. laevis proliferating nuclear cell antigen polyethylene glycol plaque forming unit(s) DNA polymerase or DNA polymerase 7 DNA polymerase 5 DNA polymerase 8 DNA polymerase III replication factor A replication factor C Saccharomyces cerevisiae single-stranded DNA binding protein sin gle-stranded DNA simian virus 40 DNA topoisomerase II Xenopus laevis xvii CHAPTER I LITERATURE REVIEW DNA REPLICATION Duplication of cellular genomes is accomplished with great efficiency and fidelity. While it is not completely understood how this process is carried out, considerable insight has been derived from studies of bacterial, bacteriophage, and viral DNA replication systems. Detailed biochemical characterization of DNA replication and its regulation is essential for our understanding of the growth and development of living organisms, and should also allow increased understanding of the perturbations of growth that occur in various disease states. Chromosomal replication in Escherichia coli (E. coli) has been studied in detail. The following general description of DNA replication draws upon our knowledge of this overall process, which appear to be evolutionarily conserved from bacteria to humans, although replication is initiated from a single origin in prokaryotes and multiple origins in eukaryotes (For reviews see Campbell, 1986; Huberman, 1987; Kelly, 1988; Kornberg, 1988; Hurwitz et al.,l990; Kornberg and Baker, 1992). The initiation of chromosomal replication requires specific DNA and protein participants. The origin of replication on duplex DNA is characterized by the presence of repeated elements serving as initiation protein binding sites, and regions rich in deoxyadenylate and thymidylate base pairs providing localized duplex unwinding sites. Initiation proteins recognize and bind the duplex origin and, with the aid of interacting proteins, promote structural conformation alteration and localized unwinding of the duplex DNA. This unwinding allows the entry of DNA helicase and single-stranded DNA binding protein (SSB), promoting further duplex unwinding and preventing reannealing and nuclease degradation of the DNA strands. Additional proteins aid the initiation process indirectly by suppressing other potential start sites to help ensure initiation at the chromosomal origin. The unwound duplex is prepared for use by replicative DNA polymerase by DNA primase which catalyzes the synthesis of an oligoodeoxyribonucleotide or —ribonucleotide serving as a primer. Because DNA polymerase can extend only a pre-existing chain, this 3 priming (which generally requires no specific DNA sequence) is always required. The association of primase with DNA polymerase or helicase activities often increases the efficiency of the events involved in starting a DNA chain. Replicative DNA polymerase extends the primer by catalyzing deoxyribonucleoside monophosphate (dNMP) incorporation complementary to the SSB coated single-stranded DNA template. This reaction involves the attack of the 3'-hydroxyl group of the primer strand on the or-phosphorous of the incoming deoxyribonucleoside triphosphate, resulting in addition of dNMP to the primer strand and the production of pyrophosphate. Replicative DNA polymerase typically utilizes accessory factors to achieve its characteristically high catalytic efficiency of dNMP incorporation and fidelity in selection of complementary moieties. The replication fork advances ahead of DNA polymerase to continually supply the enzyme with single-stranded template DNA (see figure 1). As DNA helicase opens the duplex, topoisomerases relieve the positive supercoiling induced ahead of the replication fork, and exposed single strands are coated with SSB. Replication proceeds in a semidiscontinuous manner where the leading DNA strand (the strand that is synthesized with the movement of the replication fork) is synthesized continuously, while the lagging strand (the stand that is synthesized against the movement of the replication fork) is synthesized discontinuously. In continuous DNA synthesis, DNA polymerase extends the primer continuously without need for dissociation from the primer terminus. Discontinuous DNA synthesis involves joining together small segments of DNA that are repeatedly initiated with primers. Here, a single-stranded region of template DNA must be exposed (generally 200- 1000 nucleotides), a primer is synthesized in the opposite direction of fork movement , and DNA polymerase extends this primer until it reaches the previous region of DNA (Okazaki fragment) which has been synthesized in the same way. Finally, the previously synthesized primer is removed and filled with DNA, allowing DNA ligase to seal the nick left between the extended chain and the DNA which replaced the primer. Figure l. Advancing DNA replication fork of Emil. DNA replication is toward the top of the page. As DNA helicase unwinds the duplex DNA, DNA topoisomerase relieves positive supercoiling. Sin gle-stranded DNA binding protein coats the sin gle-stranded DNA which is the template used by DNA. polymerase III holoenzyme for continuous leading strand synthesis and discontinuous lagging strand synthesis. The primosome, comprising dnaB-dnaC complex, dnaT, pri A, B, and C, and primase synthesize RNA/DNA primers for use in lagging strand synthesis. DNA polymerase I removes these primers and refills them with DNA. Ligase seals the nicks left between the pol I synthesized DNA and the DNA that is immediately 3' of it. From Kornberg and Baker (1992). 5/ Topoisomerase — PRIMOSOME ' a . ’ Ego—dnaB - dnaC complex / (La . f‘ DNA BlNDlNG 0 . ", / PRIMASE PROTEIN (SSB) 6’ DNA POLYMERASE Ill HOLOENZYME 74>, DNA \—— POLYMERASEI @«r ,4 .LIGASE -—’ 3/ 5, LEADlNG LAGGING STRAND STRAND Figure l 6 Semidiscontinuous DNA synthesis allows essentially concurrent replication of the two DNA strands; however, it requires that the priming of the lagging strand be integrated with continuous synthesis of the leading strand. A model proposed to achieve this coordination requires dimerization of DNA polymerase at the replication fork and looping of the lagging strand to endow it with the same orientation as the leading stand at the fork (Figure 2). Although events leading to the termination of DNA replication are not well elucidated, specific DNA sequences and proteins appear to be involved in bacteria. In contrast, replication of viral DNA often terminates at the meeting place of two replication forks. DNA SYNTHESIS IN ESCHERICHIA COLI Prokaryotic organisms, and E. coli in particular, provide excellent models for the study of various biochemical functions. The simplicity of chromosomal organization and the single point of initiation in prokaryotic DNA replication contribute to its usefulness as an in vitro study system. In addition, both genetic and biochemical approaches can be utilized to provide corroborative data. Replicative DNA polymerase In general, the mechanistic characteristics of replicative DNA polymerase are well suited for the function it provides in DNA replication; for a review see Young et al., 1992. DNA polymerase must catalyze rapid and highly processive DNA synthesis to ensure that replication of the entire genome is completed on the time scale of the cell cycle. Because dNMP incorporation and polymerase translocation is more rapid than incorporation, dissociation and reassociation of DNA polymerase, DNA replication is most rapid when the polymerase does not dissociate from the DNA template during the replication cycle. In addition, efficient concurrent replication of leading and lagging DNA strands requires the coordination of a dimeric DNA polymerase. Because the leading strand DNA polymerase Figure 2. Looping model for coordinated DNA synthesis of the leading and logging DNA strands. DNA replication is toward the top of the page. As the replication fork progresses, the lagging DNA strand is looped out while DNA synthesis occurs on the Okazaki fragment which was looped out in the preceeding cycle of discontinuous DNA synthesis. From O'Donnell and Studwell (1990). core \ GM! premitiotion -.- I" "I complex Figure 2 9 need not dissociate until the entire chromosome is replicated and the lagging strand polymerase must dissociate from completed products and reassociate with the next primer, these two polymerase activities must have different catalytic properties. Therefore, the polymerase complex may be capable of interacting with other proteins in the replication apparatus which alter the mechanistic characteristics of the two polymerase activities relative to each other. Finally, replicative DNA polymerase must exhibit high fidelity in nucleotide selection to ensure the viability of the daughter DNA molecules. DNA polymerase III is the replicative DNA polymerase in E. coli (For reviews see McHenry, 1991; Kornberg and Baker, 1992). DNA polymerase 111 core (pol 111 core) comprises three subunits of 130 kDa (or), 27 kDa (e), and 10 kDa (0) which are arranged in a linear conformation such that the 8 subunit modulates the interaction between the a and 0 subunits (Studwell-Vaughan and O'Donnell, 1993). The a and 8 subunits display 5'—93' DNA polymerase and 3'—)5' exonuclease activities, respectively, while the 9 subunit has no known function. However, 0 may play a role in fidelity, as its presence stimulates the proofreading exonuclease activity of the 8 subunit (Studwell-Vaughan and O'Donnell, 1993). DNA polymerase III holoenzyme (pol III holoenzyme) comprises at least seven subunits in addition to core, contributing to difficulties in the study of holoenzyme due to its instability during purification. However, the cloning, sequencing, and overexpression of all ten pol III subunits has alleviated these problems and facilitated careful studies of the individual subunits and their associated activities (Welch and McHenry, 1982; Cox and Homer, 1983; Echols et al., 1983; Scheuermann and Echols, 1984; Johanson et al., 1986; Blinkowa and Walker, 1990; Flower and McHenry, 1990; Tsuchihashi and Kornberg, 1990; Dong et al., 1993; Onrust and O'Donnell, 1993; Studwell-Vaughan and O'Donnell, 1993; Xiao et al., 1993a, b). The processivity of a DNA polymerase is simply defined as the average number of nucleotides it incorporates per polymerase-DNA association event. Processivity and the intrinsic rate of nucleotide incorporation of DNA polymerase contribute to its catalytic l 0 efficiency. The processivity of pol III core is only about 10 nucleotides (Fay et al., 1981), while that of pol III holoenzyme is greater than 5000 nucleotides and has been estimated to be as high as 150,000 nucleotides (Fay et al., 1981; Mok and Marians, 1987). Completely processive DNA synthesis of the leading DNA strand would require that pol Ill be processive for 4,700,000 nucleotides, the approximate length of the E. coli genome (Kornberg and Baker, 1992), while that of the lagging DNA strand would require a processivity of 1000-2000 nucleotides, the approximate length of an E. coli Okazaki fragment (Okazaki et al., 1973). The processivity of pol HI holoenzyme appears to be characteristic of leading strand DNA polymerase, while some subassembly of holoenzyme is likely suited for lagging strand DNA polymerase since various holoenzyme subassemblies display processivity values ranging between that of core and holoenzyme (McHenry, 1991). The propensity for replication error, or fidelity, of a DNA polymerase is typically characterized by the number of incorrect nucleotides it incorporates into the growing DNA chain. Because DNA is self propagating, uncorrected errors become permanently affixed into the genetic code; therefore, the fidelity of replicative DNA polymerase is expected to be high. Although the fidelity of pol III holoenzyme has not been carefully studied, DNA polymerase HI core misincorporates 1 nucleotide per 3 x 104 base pairs replicated (Kunkel et al., 1983). The addition of SSB lowers this error rate =37-fold to 1 misincorporation in 2 x 105 base pairs replicated (Kunkel et al., 1983). Thus, holoenzyme may display the fidelity characteristic of a replicative DNA polymerase since following DNA replication and repair errors are accumulated in vivo at a rate of 1 in 109 - 1010 base pairs replicated (Drake et al., 1969). The rate of DNA replication in E. coli has been estimated to be approximately 750 nucleotides per second in vivo (O'Donnell and Kornberg, 1985). Although pol III core is inert on natural replicative templates, holoenzyme copies these template DNAs at approximately the same rate in vitro as that observed in vivo (Godson, 1974; Bouché et al, 1 1 1975; Burgers and Kornberg, 1982a, b). Thus, DNA polymerase III holoenzyme appears to be uniquely suited as a replicative DNA polymerase in E. coli, exhibiting the catalytic efficiency and fidelity expected of an enzyme with this role. DNA polymerase accessory factors The rate, processivity, and fidelity of replicative DNA polymerase should be consistent with models of DNA replication in vivo. Although core DNA polymerase is capable of DNA synthesis, it is typicallyrless efficient than expected, given its role in vivo. Therefore, replicative DNA polymerase often associates with SSB and accessory proteins involved in enhancing processivity and primer recognition to achieve its characteristically high catalytic efficiency. Core DNA polymerase in the presence of its accessory factors is regarded as a replisome or DNA polymerase holoenzyme. Processivity factors, like the E. coli pol III B subunit, act to increase the tenacity of DNA polymerase-DNA associations. A 40 kDa monomer, the B subunit binds DNA as a dimer (Johanson and McHenry, 1980) by forming a ring around the DNA strand. X-ray crystallographic data (2.5 A resolution) has revealed that B forms an internally symmetric star-shaped ring with 12 or-helices lining the 35 A diameter inner surface and 6 B—sheets forming the 80 A diameter outer surface (Kong et al., 1992). DNA binding is achieved by virtue of this topology which allows the B subunit to slide freely along duplex DNA, acting as a sliding clamp and tethering core to DNA via its interaction with the polymerizing (0:) subunit (Stukenberg et al., 1991; Kong et al., 1992; O'Donnell et al., 1992). Thus, the B subunit can increase the processivity of DNA polymerase, but requires energy to undergo the conformational change which forms the closed ring structure encircling the DNA. This energy is provided by the primer recognition complex which chaperones the processivity factor onto primed DNA, forming the pre-initiation complex and resulting in a highly processive DNA polymerase (Wickner, 1976; O'Donnell, 1987 ; Maki and Kornberg, 1988; Onrust et al., 1991). . Five subunits of pol 11] holoenzyme compose the 1 2 primer recognition complex in E. coli: 1 (47 kDa), 5 (34 kDa), 8' (32 kDa), x (16 kDa), and w (15 kDa). The 8 and 8' subunits and x and w subunits each form separate 1:1 complexes which associate with y in a 72515'IX1W1 complex. The 7 subunit contains DNA and ATP binding activities as well as a DNA-dependent ATPase activity which is stimulated ~140-fold by 85'. Interaction of the primer recognition complex with the processivity factor occurs through the 6 subunit and stimulates the 7 subunit ATPase activity a further 3-fold (Onrust et al., 1991; Onrust and O'Donnell, 1993; Xiao et al., 1993). These activities and subunit associations led to the following model for accessory factor function (although this model seems plausible, there is little evidence regarding the order of events): the 7 subunit, in a complex with 5, 5', x, and w, binds the primed DNA template allowing 6 to guide in the B subunit. Subsequent ATP hydrolysis by the 7 subunit is coupled to assemble the ring shaped B dimer around the DNA to form the pre-initiation complex to which pol 111 core binds through its association with the B subunit (Onrust et al., 1991; Stukenberg et al., 1991; Onrust and O'Donnell, 1993). Therefore, the B subunit is a sliding clamp, activated by the 786301! complex, which tethers DNA polymerase to the DNA, conferring highly processive DNA synthesis. Although DNA polymerase III holoenzyme from E. coli catalyzes DNA synthesis at a high rate, fully efficient DNA synthesis requires SSB coated template DNA. As noted previously, the apparent general function of SSB is to coat sin gle-stranded DNA to prevent nuclease degradation and reannealing of the duplex strands. Its stimulation of DNA polymerase activity suggests that it also plays a role in rendering the substrate suitable for efficient DNA replication. E. coli single-stranded DNA binding protein is a monomer of 18.5 kDa which binds DNA as a homotetramer (Weiner et al., 1975; Williams et al., 1983; Williams et al., 1984). Mutagenesis and crosslinking studies indicate that the amino terminal third of the monomer is involved in tetramerization and DNA binding, whereas protein-protein interactions occur at the carboxyl terminus (Chase et al., 1984; Merrill et al., 1984; Williams et al., 1984; Chase et al., 1985; Griepel et al., 1987; Meyer and Laine, 1 3 1990). SSB tetramers bind single—stranded DNA (ssDNA) cooperatively, and cover a 33 nucleotide region of DNA, which likely wraps around the protein complex to utilize all four DNA binding sites (Krauss et al., 1981; Meyer and Laine, 1990). Many proteins that must bind ssDNA to carry out their functions are inhibited by SSB since they often have a lower affinity for ssDNA and cannot displace bound SSB. For example, transcription is inhibited by SSB (Niyogisk et al., 1977), as are most ssDNA nucleases (Molineux and Gefter, 1974) and DNA polymerases I and III core (Molineux et al., 1974; Fay et al., 1981, 1982). However, several enzymes involved in DNA replication, recombination and repair are stimulated by SSB: E. coli DNA helicase II (Matson and George, 1987), DNA topoisomerase I (Srivenugopal and Morris, 1986), DNA polymerase II (Bonner et al.,1988) and pol III holoenzyme (McHenry, 1988), as well as replicative DNA polymerases from bacteriophage T7 (Myers and Romano, 1988), herpes simplex virus (O'Donnell et al., 1987), and humans (Kenny et al., 1989). Whether the stimulatory capacity of SSB is due to its effects on template DNA or to direct protein- protein interactions is, in most cases, unknown. However, its ability to increase the fidelity of pol III holoenzyme is proposed to be due to its effects on template DNA conformation (Kunkel et al., 1979). The role of SSB in DNA replication appears to include some direct or indirect interaction with the other enzymes working at the replication fork in addition to coating and protecting the template ssDNA. DNA replication in E. coli must be catalyzed with high processivity, rate and fidelity to ensure chromosomal duplication within the time scale of the cell cycle. DNA polymerase core is capable of such activity on SSB coated single-stranded DNA only in the presence of its accessory factors. In fact, the catalytic parameters of DNA polymerase III holoenzyme observed in vitro are similar to those expected from analysis of mechanisms of DNA replication in vivo. This similarity implies that the enzymatic characteristics of DNA polymerases responsible for chromosomal replication in other DNA replication systems 1 4 should correlate with models of in vivo DNA replication, but may require accessory factors to do so. DNA SYNTHESIS IN BACTERIOPHAGE T4 Study of the bacteriophage T4 DNA replication apparatus has aided in, and often preceded, the understanding of analogous enzymes in E. coli. The simplicity of the T4 chromosome as well as the combined use of genetic and biochemical approaches allowed the proteins essential for T4 DNA replication fork movement to be identified, purified, and studied in vitro (Alberts et al., 1983; Cha and Alberts, 1989). Seven purified proteins encoded by the T4 chromosome generate efficient in vitro DNA elongation on nicked duplex DNA, resembling closely the in vivo T4 replication apparatus with regard to speed, geometry, and fidelity of DNA synthesis (Hubner and Alberts, 1980; Sinha et al., 1980; Nossal et al., 1983). Replicative DNA polymerase The monomeric 104 kDa protein product of bacteriophage gene 43 (gp43) is the replicative DNA polymerase of bacteriophage T4 (For a review see Young et al., 1992), exhibiting 5'—>3' DNA polymerase and 3'—)5' exonuclease activities with the respective active sites near its carboxyl and amino temrini (Sinha et al., 1980; Reha-Krantz, 1988). Bacteriophage T4 DNA polymerase in association with three additional subunits (the protein products of genes 44, 45, and 62) composes the DNA polymerase holoenzyme which exhibits a processivity of 2000—3000 nucleotides, at least 20-fold more processive than gp43 by itself (Jarvis et al., 1991). Assuming fully processive DNA synthesis, the expected processivity of leading strand DNA polymerase is 166,000 nucleotides, the approximate length of the T4 genome (Kornberg and Baker, 1992), while that of the lagging strand DNA polymerase is 1200 nucleotides, the approximate length of a T4 Okazaki fragment (Alberts et al., 1983; Selick et al., 1987. Thus, the processivity of T4 1 5 DNA polymerase holoenzyme does not appear to be characteristic for leading strand polymerase, while some subassembly of holoenzyme (or holoenzyme itself) may be well suited for lagging strand DNA polymerase. In fact, calculation of the minimum time necessary to replicate the T4 genome at the observed processivity reveals that it is longer than the latent period of wild-type T4 phage (Jarvis et al., 1991; Young et al., 1992). Although this apparent discrepancy may be reconciled by the presence of a high concentration of DNA polymerase molecules and their multiple associations with the growing DNA strand, a putative interaction between T4 DNA helicase-primase and DNA polymerase may provide an increase in processivity sufficient to do so as well (Richardson and Nossal, 1989; Young et al., 1992; Spacciapoli and Nossal, 1994a, b). T4 DNA polymerase misincorporates 1 nucleotide per 107 base pairs replicated (Loeb and Kunkel, 1982; Kunkel et al., 1984). The addition of gene products 44, 45, and 62 do not improve the fidelity of gp43 which is similar to that of T4 DNA replication in viva (Drake, 1969; Liu et al., 197 8; Sinha and Haimes, 1980) . The rate of DNA synthesis observed in vivo is approximately 500 nucleotides per second, a speed which is matched by holoenzyme in vitro (approximately 600 nucleotides per second) but not gp43 alone (250 nucleotides per second; McCarthy et al., 1976; Mace and Alberts, 1984a, b). Thus, T4 DNA polymerase holoenzyme maintains a fidelity and rate of DNA synthesis characteristic of replicative DNA polymerase, but is expected to exhibit a higher processivity to function as leading strand DNA polymerase. DNA polymerase accessory factors Bacteriophage T4 gene product 45 (gp45), a 25 kDa homodimer or homotrimer, is the processivity factor associated with T4 DNA polymerase (Spicer et al., 1982; Alberts et al., 1983; Formosa et al, 1983; Jarvis et al., 1989a; Rush et al, 1989; Reddy et al., 1993). This processivity factor has been shown by crosslinking and DNA footprinting analyses to bind DNA alone or in a complex with the primer recognition complex (see below; l 6 Hockensmith et al., 1986; Capson et al., 1991; Munn and Alberts, 1991a), and is hypothesized to form a homotrimeric ring around duplex DNA, similar to the "sliding clamp" formed by the B subunit of E. coli DNA polymerase III (Alberts, 1984; Jarvis et al., 1989; O'Donnell and Studwell, 1990; Gogol et al., 1992). While gp45 exhibits no obvious amino acid sequence similarity to pol III B subunit, comparison of the gp45 predicted secondary structure with the tertiary structure of pol III B subunit derived from x- ray crystallographic studies does reveal a weak structural alignment (Kong et al., 1992). A more detailed comparison will depend on the determination of the three dimensional structure of T4 gp45. As in E. coli, the primer recognition complex appears to provide the energy required to lock the processivity factor onto the DNA. The products of bacteriophage T4 genes 44 and 62 (gp44 and gp62) are 36 kDa and 21 kDa respectively, and exist as a tightly bound complex of four gp44 to one gp62 (gp44/62; Spicer et al., 1984; Jarvis et al., 1989a; Rush et al., 1989). Like the E. coli primer recognition complex, the gp44/62 complex exhibits an ATPase activity which is stimulated ~20-fold by DNA and a further =100-fold by the T4 processivity factor (Mace and Alberts, 1984a; Jarvis et al., 1989b; Rush et al, 1989). Although the ATPase active site is located in gp44 (similar to E. coli 7 subunit, to which it exhibits considerable homology), stimulation of this activity by gp45 is only slight in the absence of gp62, suggesting a role for gp62 as a mediator for the interaction between gp44 and gp45 (similar to E. coli 8 subunit; Rush et al., 1989; O'Donnell et al., 1993). The mode of action of T4 DNA polymerase and its accessory factors is likely to be similar to that of its E. coli counterparts, although direct proof has not yet been obtained. Assuming analogy to the model proposed for E. coli, the primer recognition complex (gp44/62) binds the primed template, allowing gp62 to guide in the processivity factor (gp45). Subsequent ATP hydrolysis by gp44 is coupledto assemble the ring shaped gp45 trimer around the DNA to form the preinitiation complex to which T4 DNA polymerase l 7 binds through its association with gp45. Thus, gp45 appears to be a sliding clamp activated by the gp44/62 complex which tethers DNA polymerase to the DNA and confers upon it high processivity. Fully efficient synthesis in bacteriophage T4 DNA replication, as in E. coli DNA replication, requires protein-coated DNA template. Bacteriophage single—stranded DNA binding protein, the product of the T4 gene 32 (gp32), is a 33.5 kDa monomer in dilute solution (Carroll et al., 1975; Kirsch and Allet, 1982), but dimer- and trimerizes at estimated physiological concentrations (von I-Iippel et al., 1982). Proteolysis experiments indicate that, as for E. coli SSB, this subunit interaction occurs through the arrrino terminal amino acids (Spicer et al., 1979; Williams et al., 1979; Hosoda et al., 1980; Lonberg et al., 1981; Williams and Konigsberg, 1981), while gp32 association with other proteins (see below) occurs through the carboxyl terminal amino acids (Burke et al., 1980; Chase and Williams, 1986). The DNA binding domain of gp32 is suspected to reside in the central region of the protein (Williams et al., 1980, 1981; Williams and Konigsburg, 1981; Chase and Williams, 1986), through which it binds cooperatively to ssDNA to an area spanning eight nucleotides (Alberts and Frey, 1970; Jensen et al., 1976; Newport et al., 1981; Prigodich et al., 1984). Although they are clearly functional homologs, sequence comparison of T4 gp32 and E. coli SSB reveals very little homology (Sancar et al., 1981; Williams et al., 1983). Affinity chromatography indicates that T4 gp32 associates physically with both the T4 DNA polymerase and processivity factor, as well as several proteins involved in DNA recombination (Formosa et al., 1983). In addition, gp32 stimulates DNA polymerase core activity 5- to 10-fold (Huberman et al., 1971; Nossal and Peterlin, 1979), holoenzyme activity 100- to ZOO-fold (Nossal and Peterlin, 1979; Alberts et al., 1980), and increases DNA polymerase fidelity 15- to 20-fold, likely by increasing nucleotide specificity (Gillen and Nossal, 1976; Topal, and Sinha, 1983). Binding of gp32 to single-stranded DNA at the T4 replication fork, like binding of SSB at the E. coli replication fork, prevents 1 8 nuclease degradation and reannealing of duplex DNA. Its stimulation of DNA polymerase activity suggests that it also plays a role in rendering the substrate suitable for efficient DNA replication. E. coli SSB cannot substitute for gp32 in stimulation of T4 DNA polymerase activity or fidelity, suggesting that the two coating proteins impose different conformations onto the bound DNA or that direct protein-protein interactions are very important, or both (Sigal et al.,1972; Burke et al., 1980). Bacteriophage T4 DNA polymerase utilizes accessory factors similar to those used by E. coli DNA polymerase III to catalyze efficient DNA synthesis. Although as yet unproven, the mechanisms employed by these accessory factors appear conserved between the two systems. These similarities support the hypothesis that DNA polymerases responsible for chromosomal replication in various DNA replication systems may require accessory factors to allow correlation of their enzymatic characteristics with models of in vivo DNA replication. DNA SYNTHESIS IN SIMIAN VIRUS 40 The study of replication of eukaryotic genomes is complicated by initiation of DNA replication at multiple sites. Therefore, models of single initiation sites are studied and the results applied to overall replication events. Simian virus 40 (SV40) DNA replication occurs in the host (human) cell nucleus and represents a model of a single replicon in eukaryotic DNA replication (For a review see Kelly, 1988). A double-stranded circular 5000 base pair molecule, the viral genome is complexed with histones to form nucleoprotein structures similar to cellular chromatin and encodes only one protein required for its replication. The remainder of the DNA replication machinery, including DNA polymerase and accessory factors, is provided by the host cell. l 9 Multiple DNA polymerases in eukaryotic DNA replication While prokaryotic and bacteriophage genomes are copied by single DNA polymerases, eukaryotic genomes employ at least three DNA polymerases for efficient DNA replication. In contrast, E. coli utilizes three DNA polymerases in cellular function, but only DNA polymerase III is a replicative enzyme. DNA polymerase I is involved in DNA repair and gap filling, while DNA polymerase 11 appears to have a role in repair of damaged DNA (Tait et al., 1974; Bonner et al., 1988). Biochemical analysis of SV40 DNA replication in vitro indicates that DNA polymerases or (pol or) and 8 (pol 8) are required to copy the viral genome efficiently (Murakami et al., 1986; Lee et al., 1989; Weinberg and Kelly, 1989). Further, genetic studies have revealed that DNA polymerases a, 8, and c (pol e) are required for replication of yeast DNA in vivo (Johnson et al., 1985; Morrison et al., 1990). The lack of a polymerase requirement in SV40 DNA replication in vitro appears to indicate that either the replication of the viral genome differs from cellular chromosomal DNA replication (Linn, 1991), or that pol 8 can substitute for pol e in vitro. Nevertheless, considerable insight into the proteins involved in eukaryotic DNA replication has been gained through the study of the SV40 system. Biochemical characterization of DNA polymerases a and 8 seemed to indicate that they were suited for lagging and leading strand DNA synthesis, respectively, in SV40 DNA replication. However, the requirement of the processivity factor associated with pol 8 in both leading and lagging strand DNA synthesis implicated pol 8 as the sole replicative DNA polymerase (Downey et al., 1988; Prelich and Stillman, 1988; Bullock et al., 1991), leading to a hypothesized role for pol or in priming of DNA synthesis on both DNA strands with, perhaps, some limited role in primer extension. In yeast, pol 8 was assigned the role of lagging strand DNA polymerase due to its conditional processivity while the highly processive pol e was designated the leading strand DNA polymerase (Morrison et al., 1990). However, the data presently available does not allow conclusive assignment of DNA polymerases or, 8, or e as leading or lagging strand DNA polymerases. For the 2 O purposes of this report the prevailing opinions in the eukaryotic DNA replication literature will be observed: pol 8 is the leading strand DNA polymerase, pol e is the lagging strand DNA polymerase, and pol 0t primes DNA synthesis and partially extends primers for use by pols 8 and 8. Leading strand DNA polymerase DNA polymerase 8 is a heterodimer comprising 125 kDa and 50 kDa subunits and exhibiting 5'—>3' DNA polymerase and 3'—->5' exonuclease activities (Boulet et al., 1989; Downey et al., 1990; Syvaoja et al., 1990). The large subunit is the catalytic subunit while the function of the small subunit is unknown, but may mediate the interaction between the DNA polymerase and its processivity factor (Goulian et al., 1990; Syvaoja et al., 1990). DNA polymerase 8 holoenzyme comprises pol 8 and its processivity (proliferating cell nuclear antigen; PCNA) and primer recognition (replication factor C; RF-C) factors. The cDNAs encoding PCNA and the subunits of RF-C have been identified, sequenced and, in some cases, overexpressed, facilitating their study (Almendral et al, 1987; Chen et al., 1992a,b; Burbelo et al., 1993; O'Donnell et al., 1993; Howell et al., 1994). The processivity of DNA polymerase 8 core is 5 to 25 nucleotides, while that of pol 8 in the presence of PCNA is greater than 2000 nucleotides (Crute et al., 1986; Prelich et al., 1987b; Sabatino et al., 1988; Syvaoja et al., 1990). Presumably, the processivity of 8 polymerase in the presence of both PCNA and RF-C would be even higher. The expected processivity of a completely processive leading strand DNA polymerase is 5000 nucleotides in SV40, the length of the SV40 genome, but is 10,000 to 300,000 nucleotides in chromosomal DNA replication, the approximate range in lengths of single eukaryotic replicons (Kornberg and Baker, 1992). Processive lagging strand DNA replication requires a processivity of 135 to 250 nucleotides, the approximate length of an Okazaki fragment in eukaryotes (Anderson and DePamphilis, 1979; Nathanel and Kaufmann, 1990). 2 1 Although the fidelity of 8 polymerase core cannot be determined due to its inefficient use of single—stranded DNA, the accuracy of pol 8 in the presence of PCNA has been estimated to be at least 3-fold lower than that observed in vivo (Thomas et al., 1991). While pol 8 holoenzyme polymerizes DNA at a speed of 110 nucleotides per second in vitro, DNA polymerase 8 core synthesizes DNA at a rate of only 2 nucleotides per second (Burgers, 1991). In comparison, the rate of replication fork movement in eukaryotes in vivo is estimated to be between 50 and 60 nucleotides per second (Blumenthal et al., 1974; Rivin and Fangman, 1980). Thus, DNA polymerase 8 holoenzyme exhibits a rate of DNA synthesis characteristic of replicative DNA polymerase and may exhibit processivity and fidelity values consistent with this role as well. DNA polymerase accessory factors The processivity factor associated withDNA polymerase 8 (PCNA) is a 36 kDa homotrimer which is required for efficient SV40 DNA replication in vitro (Bravo et al., 1987; Prelich et al., 1987a; Bauer and Burgers, 1988). PCNA promotes binding of pol 8 to the template-primer (N g et al., 1993), stimulates pol 8 activity 20- to lOO-fold ('T an et al., 1986; Bauer and Burgers, 1988; Ng et al., 1990) and processivity approximately 10- fold (Kunkel et al., 1990), yet has no effect on or polymerase and little effect on e polymerase (Hamatake et al., 1990). PCNA, like T4 gp45, is hypothesized to form a homotrimeric ring around duplex DNA, similar to the "sliding clamp" formed by the B subunit of E. coli DNA polymerase III. While PCNA, again like T4 gp45, exhibits no obvious amino acid sequence similarity to pol III B subunit, comparison of the predicted secondary structure for PCNA with the tertiary structure of pol III B subunit derived from x-ray crystallographic studies does reveal a weak structural alignment (Kong et al., 1992). In contrast, sequence comparison of PCNA to T4 gp45 reveals 30% to 50% arrrino acid similarity between the two processivity factors (T surimoto and Stillman, 1990), which are 2 2 likely more structurally similar to each other than to E. coli B subunit given their hypothesized trimeric binding conformation. As in E. coli and bacteriophage T4, the primer recognition complex appears to provide the energy required to lock the processivity factor onto the DNA. Replication factor C comprises a large subunit of approximately 140 kDa, containing DNA binding activity, and four small subunits of approximately 40 kDa (Tsurimoto and Stillman, 1990; Lee et al., 1991; Tsurimoto and Stillman, 1991; Yoder and Burgers, 1991; Fien and Stillman, 1992). One of the small subunits contains an ATPase activity which, similar to the A’I'Pase activities of E. coli and T4 primer recognition complexes, is stimulated ~20- fold by DNA and a further =4-fold by its homologous processivity factor, suggestive of a PCNA:RF-C interaction (Tsurimoto and Stillman, 1990, 1991; Yoder and Burgers, 1991; Chen et al., 1992a). In addition, RF-C appears to be analogous to other primer recognition complexes in its ability to facilitate the binding of PCNA to template-primer (T surimoto and Stillman, 1991). The genes for all of the subunits of RFC have been identified and sequenced. The large (140 kDa) subunit of RF-C exhibits homology to the small subunits (27% identical and 48% similar over 186 amino acids), which exhibit homology to each other, to T4 gp44 (=30% amino acid identity and =65% similarity), and to the E. coli 7 subunit (=20% amino acid identity and ~70% similarity; Chen et al., 1992 a, b; O'Donnell et al., 1993). Crosslinking and photoaffinity labelling experiments indicate that in RF-C, only the 40 kDa subunit contains ATP binding activity even though the 140, 40, 37 and 36 kDa subunits, as well as T4 gp44 share an ATP binding motif which is nearly identical to the motif found in the E. coli 7 subunit (Spicer et al., 1984; Chen et al., 1992a, b; Burbello et al., 1993; O'Donnell et al., 1993; Howell et al., 1994). The high degree of similarity of replication factor C with primer recognition complexes from T4 and E .coli provide support for its hypothesized analogous role in eukaryotic DNA replication. 2 3 The mode of action of DNA polymerase 8 and its accessory factors is likely to be similar to that of its bacteriophage T4 and E. coli counterparts, although direct proof has not yet been obtained. Assuming analogy to the model proposed for E. coli, the primer recognition complex, replication factor C, binds the primed template through action of its 140 kDa subunit, thereby permitting association of the processivity factor, PCNA. Subsequent ATP hydrolysis by RF—C is coupled to assemble the ring shaped PCNA trimer around the DNA to form the preinitiation complex to which pol 8 binds through the putative association of its small subunit with PCNA (Hurwitz et al., 1990). Therefore, PCNA appears to be a sliding clamp activated by the RFC complex which tethers DNA polymerase to the DNA, facilitating processive DNA synthesis. Lagging strand DNA polymerase DNA polymerase 8 or 8 appear capable to serve as either the leading or lagging strand DNA polymerase. However, because pol e is not required in SV40 DNA replication in vitro (Murakami et al., 1986; Lee et al., 1989; Weinberg and Kelly, 1989), it has been relegated to the role of lagging strand DNA synthesis in eukaryotic DNA replication in general. DNA polymerase 8 comprises a large catalytic subunit of 200-260 kDa and possibly two additional subunits of 80 and 34 kDa (Budd et al., 1989; Syvaoja and Linn, 1989; Hamatake et al., 1990; Syvaoja et al., 1990). Both 5'-)3‘ DNA polymerase and 3'->5' exonuclease activities are harbored by a polymerase which is stimulated ~7-fold by PCNA and RF-C, but is unaffected by either accessory factor acting on its own (Goslin and Bymes, 1982; Lee et al., 1984; Bauer et al., 1988). The processivity of pol e is greater than 1500 nucleotides and is unaffected by PCNA, although PCNA and RF-C together may alter it (Syvaoja and Linn, 1989; Syvaoja et al., 1990). This high processivity does not appear to be well suited for Okazaki fragment synthesis of only 135- 250 nucleotides (Anderson and DePamphilis, 1979; Nathanel and Kaufmann, 1990). DNA polymerase e misincorporates 1 nucleotide per 2 x 105 incorporated, an error rate 2 4 approximately 3-fold higher than that observed in vivo and similar to that of pol 8 in the presence of PCNA (Kunkel et al., 1987; Thomas et al., 1991). The in vivo rate of replication fork movement of 50 to 60 nucleotides per second is matched by pol e in the presence of PCNA and RF-C, but pol 8 alone incorporates only 7 nucleotides per second (Blumenthal et al., 1974; Rivin and Fangman, 1980; Burgers, 1991). Thus, only the rate of DNA synthesis and perhaps the fidelity of nucleotide selection by pol 8 appears suited for its putative role as a lagging strand DNA polymerase. The inherent high processivity of pol 8 seems to favor a role in leading DNA strand synthesis, as suggested by Morrison and colleagues (1990). In fact, the low processivity of pol 8 core implicates it, or a subassembly of pol 8 holoenzyme, in lagging strand DNA synthesis; whereas, the PCNA requirement of leading strand DNA synthesis of SV40 DNA replication in vitro may reflect the ability of pol 8 to substitute for pol e in this system. DNA polymerase—primase DNA polymerase a appears to initiate leading strand DNA synthesis at the replication origin, prime Okazaki fragment synthesis on the lagging DNA strand, and catalyze some limited extension of these primers (Morrison et al., 1990). Required in SV40 DNA replication in vitro, pol or comprises four subunits including a 140-185 kDa subunit containing DNA polymerase activity, a 70 kDa subunit of unknown function, and 60 and 50 kDa subunits exhibiting DNA primase activity (Kaguni et al., 1983; Plevani et al., 1985; Suzuki et al., 1985; Murakami et al., 1986; Lee et al., 1989; Weinberg and Kelly, 1989). DNA primase synthesizes primers 24-28 nucleotides in length and is either unaffected or inhibited by the presence of DNA polymerase accessory factors (Conaway and Lehman, 1982; Badarocco et al., 1985; Singh et al., 1986; Cotterill et al., 1987). The association of DNA polymerase with primase limits the primer length to 12-14 nucleotides and appears to suppress multimeric primer synthesis (Conaway and Lehman, 1982; Badaracco et al., 1985). 2 5 DNA polymerase-primase is normally processive for 8-20 nucleotides, but manipulation of reaction conditions promotes processive DNA synthesis of up to 1000 nucleotides (Villani et al., 6., 1981; Pritchard et al., 1983; Cotterill et al., 1987; Sabatino et al., 1988). Depending on the source of pol a, the polymerase misincorporates 1 nucleotide per 2 x 104 to 4 x 105 incorporated, 2- to 35-fold lower than the fidelity observed in vivo (Kaguni et al., 1984; Kunkel etal., 1987; Thomas etal., 1991‘). ,Fmally, the rate of DNA synthesis by pol or is approximately 2 nucleotides per second, 25- to 30- fold slower than observed fork movement in vivo (Blumenthal et al., 1974; Banks et al., 1979; Rivin and Fangman, 1980; Hubscher et al., 1982). Thus, the enzymatic characteristics of DNA polymerase or do not correlate well with that of a replicative enzyme, but appear to suit its hypothesized role of priming and limited primer extension. In summary, DNA polymerase or appears to be responsible for priming both the leading and lagging DNA strands, while polymerases 8 and a likely carry out the bulk of extension of these primers. It is unclear whether pol 8 or pol e is involved in leading strand DNA replication. DNA polymerase accessory factors appear to have the largest influence over pol 8; therefore, determination of the specific involvement of these accessory factors in DNA replication may allow more defined elucidation of the role of pol 8 in vivo. Single-stranded DNA binding protein Eukaryotic single-stranded DNA binding protein (replication factor A; RF-A) is structurally dissimilar, but appears to be functionally analogous, to E. coli SSB and bacteriophage T4 gp32. Replication factor A coats single-stranded DNA, preventing nuclease degradation and reannealing of duplex, and is required for SV40 DNA replication in vitro (Fairman and Stillman, 1988). A heterotrimer comprising 70 kDa, 30 kDa, and 14 kDa subunits, RF—A binds DNA with low cooperativity and a site size of 22 nucleotides (Wobbe et al., 1987; Fairman and Stillman, 1988; Wold and Kelly, 1988; Mitsis et al., 1993). DNA binding activity resides in the 70 kDa subunit (Wold et al., 1989), while the 2 6 30 kDa and 14 kDa subunits may be involved in protein-protein interactions (Erdile et al., 1990; Umbricht et al., 1993). RF—A interacts directly with pol a-primase and the SV40 T- antigen, and stimulates their polymerase and helicase activities, respectively, as well as the polymerase activity of pol 8 (Stahl et al., 1986; Dean et al., 1987; Wold et al., 1987; Kenny et al., 1989; Tsurimoto and Stillman, 1989a; Domreiter et al., 1992). Thus, in addition to its DNA coating function, RF-A appears to aid in promoting replication origin unwinding and priming by mediating the interaction between T-antigen (origin recognition and helicase activities) and pol or (priming activity; Melendy and Stillman, 1993). Eukaryotic DNA replication, as modeled by SV40 DNA replication, is similar to prokaryotic and bacteriophage DNA replication in many respects. Semidiscontinuous DNA replication is carried out by a pair of DNA polymerases possessing differing catalytic properties. In E. coli and bacteriophage T4, replicative DNA polymerase appears to dimerize and the leading and lagging strand DNA polymerases achieve altered enzymatic characteristics by virtue of each monomer's association with different accessory factors, while in SV40 DNA replication two different DNA polymerase assemblies appear to be utilized. Rapid and processive synthesis catalyzed by leading strand DNA polymerase requires the presence of polymerase accessory factors which appear to be functionally conserved from bacteria to humans. Although no obvious amino acid sequence homology is shared between the bacterial processivity factor and its bacteriophage and eukaryotic homologs, they do appear to be functionally and structurally conserved. In addition, bacteriophage and eukaryotic processivity factors exhibit some amino acid sequence similarity, as do primer recognition factors from bacteria, bacteriophage and humans. Finally, single-stranded DNA binding proteins from the three systems are clearly functionally similar, although they share no amino acid sequence homology. In summary, DNA polymerases throughout widely divergent DNA replication systems exhibit similar mechanisms of DNA synthesis and require accessory factors for efficient catalysis. In addition, the mechanistic characteristics of replicative DNA 2 7 polymerases are well suited for the function they provide in DNA replication. Thus, the correlation of the enzymatic characteristics of DNA polymerase with models of DNA replication appear to be indicative of either the lack of required polymerase accessory factors or the presence of additional DNA polymerases which are enzymatically more well Suited for a particular cellular function. MITOCHONDRIAL DNA REPLICATION As energy producing organelles, mitochondria are required in virtually all eukaryotic cells. Mitochondrial function is dependent on the expression of its genome which encodes thirteen polypeptides required in electron transport and oxidative phosphorylation, two ribosomal RNAS, and twenty-two transfer RNAS (Tzagaloff and Myers, 1988). Mitochondrial DNA mutations can lead to disease and have been proposed to contribute to aging and common degenerative diseases including epilepsy, cardiac disease, Parkinson's disease, and Alzheimer's disease, as well as maturity onset diabetes (Wallace, 1992). Many mutations in mitochondrial DNA are associated with ophthalmological and neurological disease. Disease resulting from point mutations in protein encoding sequences are most commonly found associated with mutations in NADH dehydrogenase subunits 1, 2, 4, and 5, cytochrome b, and ATP synthase subunit 6 genes (Singh et al., 1989; Holt et al., 1990; Howell et al., 1991a, b; Huoponen et al., 1991; Johns and Berman, 1991; Brown et al., 1992). Conditions resulting from point mutations in tRNA genes are most commonly associated with defects in complex I (NADH dehydrogenase complex) and IV (cytochrome oxidase complex), the two complexes for which mtDNA encodes the largest number of subunits (Shoffner et al., 1988; Goto et al., 1990; Hammans et al., 1991; Hess et al., 1991; Inc et al., 1991; Johns and Hurko, 1991; Lauber et al., 1991; Yoon et al, 1991). Deletions of mtDNA typically occur between the origin of replication and the initiation site of lagging strand DNA synthesis and are often flanked by 13 nucleotide long 2 8 direct repeats (Wallace, 1989; Wallace et al., 1991). Although there are several proposed mechanisms for the appearance of these deletions, the Slip-replication mechanism appears to be the favored model (Shoffner et al., 1989; Mita et al., 1990). In this model, while the lagging strand is displaced during DNA replication, an upstream repeat on the lagging strand base pairs with a downstream repeat on the leading strand. A break in the lagging strand downstream of the base paired repeats serves as a primer for DNA synthesis, resulting in deletion of the region between the direct repeats. This slip-replication model has also been implicated in the proposed relationship between mitochondrial DNA damage and aging. Oxidative phosphorylation normally declines with age, apparently due to a time dependent increase in mtDNA damage caused by oxygen radicals (Linnane et al., 1989;; Cortopassi and Amheim, 1990; Corral-Debrinski et al., 1991). This damage leads to inhibition of replication and transcription which, by prolonging the time the DNA is triple stranded, fosters mitochondrial DNA deletions. Because the rate of mtDNA replication appears to be directly proportional to its length, deleted DNAS have a replicative advantage over normal DNAS and become enriched with time (Larsson et al., 1990). Elucidation of the mechanisms involved in the faithful duplication of the mitochondrial genome may provide us with a more detailed understanding of these genetic disorders. Vertebrate mitochondrial DNA is a double-stranded circular molecule, ranging in size from 16.5-17.6 Kb (Gray, 1989). A single substantial non-coding region contains the origin of replication (Brown and Vinograd, 1974; Robberson et al., 1974; Tapper and Clayton, 1981) and promoters for transcription of both DNA strands (Figure 3; Clayton, 1991). Replication of vertebrate mitochondrial DNA appears to be initiated by RNA polymerase which synthesizes transcripts for use as primers for DNA synthesis (Chang and Clayton, 1985; Chang et al., 1989). Therefore, the initiation of DNA replication is described by the initiation of transcription at the light strand promoter since the light strand is the template for leading DNA strand synthesis. The light strand promoter is an approximately 50 base pair sequence located about 100 base pairs upstream of the origin of 29 Figure 3. Representation of vertebrate mitochondrial DNA. The shaded areas represent the 22 tRNA genes. The 128 and 168 rRNA genes and non-coding region (D-loop) are shown. 0 and I are the origin of replication and initiation site of lagging strand DNA synthesis, respectively. LSP and HSP are the respective promoters for transcripts copied from the leading and lagging DNA template strands. Arrows denote the direction of synthesis. The protein coding genes are: cytochrome c oxidase subunits I, II, and III (COI, COII, COIII); ATPase subunits 6 and 8 (ATP6, ATP8); cytochrome b (CYT b); and subunits l, 2, 3, 4, 4L, 5, and 6 of NADH dehydrogenase (NDl, ND2, ND3, ND4, ND4L, NDS, ND6). From Clayton (1991). 30 Figure 3 3 1 DNA replication (Chang et al., 1987; Topper and Clayton, 1989). It comprises two functionally distinct domains: a short sequence near the'transcriptional start site, and a sequence 25-35 base pairs upstream which is required to stimulate transcription (Chang and Clayton, 1984; Hixson and Clayton, 1985; Chang and Clayton, 1986a, b). This stimulation also requires the binding of a putative DNA packaging protein, mtTFA (Fisher et al., 1987; Fisher and Clayton, 1988; Parisi and Clayton, 1991; Parisi et al., 1993), and possibly an additional factor to ensure promoter specificity (Fisher et al., 1989). The transition of RNA priming to DNA synthesis occurs over a region of three short conserved sequence elements (Walberg and Clayton, 1981; Chang and Clayton, 1985; Chang et al., 1985) and may involve a ribonucleolytic mitochondrial RNA processing protein whose role is a subject of controversy: although this protein cleaves RNA transcripts between two of the three conserved sequences, the locations of these cleavage sites do not correlate exactly with the locations of the majority of RNA to DNA transitions (Chang and Clayton, 1987a). Further, the RNA component of the processing ribonucleoprotein is much more abundant in the nucleus than the mitochondria and appears to play a role in the processing of pre 5.8S ribosomal RNA in the nucleus (Chang and Clayton, 1987b; Schmitt and Clayton, 1993). Biochemical and electron microscopic analyses of replication intermediates reveal that mitochondrial DNA synthesis is unidirectional and asymmetric (Figure 4), where synthesis of the lagging DNA strand is not initiated until synthesis of the leading DNA Strand is 67% complete (Matsumoto et al., 1974). This asymmetry is due to the distant location of the lagging strand initiation site which must be exposed in single-stranded form by leading strand DNA synthesis in order to function (Wong and Clayton, 1985). In vertebrate mtDNA, this initiation site is an approximately 30 nucleotide region surrounded by several tRNA genes (Martens and Clayton, 1979) which, upon exposure, is capable of forming a stem-loop structure hypothesized to be required for its recognition. Primer formation is complementary to the T-rich loop and is proposed to be catalyzed by 32 Figure 4. Model of mouse mitochondrial DNA replication. The order of replication is clockwise starting with (A). O and I are the origin of replication and initiation site of lagging strand DNA synthesis, respectively. The double arrows between (A) and (G) reflect the metabolic instability of the D-loop strands and the consequent equilibrium between superhelical mtDNA (G) and the D-loop DNA (A). DNA synthesis from the replication origin is depicted prior to (B) and after the initiation of lagging strand DNA synthesis (C) and (D). Both daughter molecules (E) are converted to closed circles (F) which are negatively supercoiled (G) to form the substrate for D-loop strand synthesis. From Clayton (1982). 33 A V O Figure 4 3 4 mitochondrial DNA primase, while the RNA/DNA primer to DNA transition occurs near the base of the stem (Wong and Clayton, 1985a, b). Subsequent elongation of the lagging DNA strand appears also to be continuous. Thus, mitochondrial DNA replication may proceed in a fully continuous and processive fashion with no requirement for intricate looping models coordinating synthesis of the two DNA Strands. The resultant products of mtDNA replication are two interlocked DNA molecules which are segregated and converted to closed circles into which superhelical turns are introduced (Berk and Clayton, 1974; Bogenhagen and Clayton, 1978). Drosophila mitochondrial DNA replication appears to be generally similar to that of vertebrate mtDNA. Drosophila, like vertebrates, harbor a double-stranded circular genome ranging in size from 15.7 to 19.5 Kb (Fauron and Wolstenholme, 1976). A single non- coding region contains the origin of replication and appears to contain the initiation site for lagging Strand DNA replication as well (Goddard and Wolstenholme, 1978, 1980). This non-coding region (A+T region) is composed of 90-96% deoxyadenylate and thymidylate residues (Clary and Wolstenholme, 1985, 1987; Monnerot et al., 1990; Lewis et al., 1994) exhibiting no sequence similarities to its vertebrate mtDNA counterpart (Clary and Wolstenholme, 1985; Lewis et al., 1994). Although the specifics of initiation are unknown, Drosophila mtDNA replication originates within the A+T region and proceeds in a unidirectional and asymmetric manner (Figure 5) where leading strand synthesis is up to 99% complete prior to initiation of lagging strand synthesis (Wolstenholme et al., 1979). Drosophila embryos seem to be a good source of mitochondrial DNA replication proteins, and the study of replication catalyzed by these proteins is valuable as a model for that catalyzed in mammalian systems. In addition, the Study of these proteins and processes may enhance comparative analyses between lower eukaryotes, insects and mammalians. Insects are both valuable and pestilent organisms as they carry out many pollinating activities, serve as food for other animals, and conduct valuable scavenger services, but also are responsible for costly crop loss and may disrupt the health of humans 35 Figure 5. Model of Drosophila mitochondrial DNA replication. In (a), the origin of replication, 0, and the direction of replication, R, around the molecule are indicated. DNA synthesis from the replication origin is depicted prior to (b) and (c), and after the initiation of lagging strand DNA synthesis (d). Both daughter molecules (e) and (f) are converted to closed circular DNAS. Modified from Wolstenholme and colleagues (1979). 36 DROSOPHILA MITOCHONDRIAL DNA REPLICATION Figure 5 3 7 and other mammals. Their ability to breed rapidly and produce a large number of offspring, as well as the ease with which they are raised contributes to the value of Drosophila as biochemical and genetic insect models. Mitochondrial RNA polymerase and DNA primase Mitochondrial RNA polymerase core is a 140-145 kDa monomer which possesses ribonucleotide polymerizing activity, but requires additional subunits for in vitro initiation at promoter sequences (Kelly and Lehman, 1986; Bogenhagen and Insdorf, 1988). Encoded by a nuclear gene which has been cloned from S. cerevisiae, core RNA polymerase has been identified and purified from both S. cerevisiae and X. laevis tissues (Kelly and Lehman, 1986; Bogenhagen and Insdorf, 1988). RNA polymerase holoenzyme, which forms on DNA but not in solution, comprises core and a 43 kDa specificity factor which displays characteristics similar to those of bacterial sigma factors (Jang and J aehning, 1991). In X. laevis, this factor binds an eight nucleotide region which surrounds the transcription initiation site and is required for specific initiation of transcription (Bogenhagen and Insdorf, 1988; Bogenhagen and Romanelli, 1988). RNA polymerase holoenzyme has been implicated in the events leading to initiation of DNA replication even though it cannot incorporate dNMPs, a common feature of DNA primase, and does not synthesize exclusively short RNA chains (Winkley et al., 1985; Kelly and Lehman, 1986). Mitochondrial DNA primase appears to be involved in the priming of lagging strand DNA synthesis, and has been partially purified from human and rat tissues (Wong and Clayton, 1985b; Ledwith et al., 1986). Products synthesized complementary to the lagging DNA strand initiation site by a protein fraction containing human mtDNA primase and mtDNA polymerase were characterized by the presence of a short (9-12 nucleotide) stretch of RNA covalently linked to greater than 100 nucleotides of DNA (Wong and Clayton, 1985a, b). Similarly, Xenopus mtDNA primase catalyzed the formation of 1-12 nucleotide 3 8 long RNA primers but also synthesized 25-40 nucleotide stretches of DNA (Ledwith et al., 1986), consistent with the production of mixed RNA/DNA primers characteristic of DNA primase activities. Mitochondrial DNA topoisomerase Because mitochondrial DNA is a closed, circular and topologically constrained molecule, its replication likely requires the presence of topoisomerase to relax the positive supercoils introduced into it by DNA unwinding. Further, upon completion of replication, resolution of daughter DNA molecules and their negative supercoiling likely occurs through the action of topoisomerase. Mitochondrial DNA topoisomerase I appears to fill the former role, as it removes positive supercoils (Fairfield et al., 1979; Callen, 1981; Lazarus et a1. 1987) and inhibitor studies suggest that it is involved in mtDNA replication and transcription (Fairfield et al., 1985). In addition, its putative association with yeast ABF2 (analogous to human mtTFA) appears to induce a DNA gyrase activity which might be expected to introduce negative supercoils into daughter DNA molecules (Caron et al., 1979; Diffley and Stillman, 1991). In contrast, mitochondrial DNA topoisomerase H appears to play a role in the decatenation of kinetoplast DNA in trypanosome mitochondria as well as the relaxation of negatively supercoiled DNA (Melendy and Ray, 1989). Mitochondrial DNA helicase A mitochondrial DNA helicase identified in S. cerevisiae is an ATP dependent enzyme which unwinds DNA in the 5'—>3' direction (Lahaye et al., 1991) and appears to be involved in mitochondrial DNA repair through recombination (Foury and Kolodynski, 1983). Although genetic analyses indicate that it is not essential to the replication of mtDNA, it may participate in replication events by eliminating local secondary structure (Foury and Lahaye, 1987) similar to E. coli DNA helicase II. In fact, yeast mtDNA helicase exhibits homology to this E. coli DNA helicase (Foury and Lahaye, 1987) which 3 9 plays a role in DNA excision repair and recombination (Kornberg and Baker, 1992). In contrast, a DNA helicase identified in bovine mitochondria exhibits 3'—>5' unwinding activity (Hehman and Hauswirth, 1992), a characteristic Similar to E. coli Rep helicase which is essential in rolling circle DNA replication that some viral DNAS undergo (Kornberg and Baker, 1992). This mechanism of replication is similar to leading strand DNA synthesis in mitochondrial DNA replication. Replicative DNA polymerase In general, the mechanistic characteristics of mitochondrial DNA polymerase are expected to be Similar to those of its chromosomal counterparts. That is, DNA polymerase should catalyze rapid and highly processive DNA synthesis as well as exhibit a high fidelity of nucleotide selection to ensure the viability of the daughter DNA molecules. However, unlike chromosomal DNA polymerases, the coordination of a dimeric polymerase for concurrent replication of leading and lagging DNA strand is not required. In addition, because neither the leading strand nor the lagging strand DNA polymerase need dissociate until the entire mitochondrial genome is replicated, mitochondrial DNA polymerase may be expected to catalyze only highly processive DNA synthesis, as is characteristic of chromosomal leading Strand DNA polymerases. Animal mitochondrial DNA polymerase, DNA polymerase 'y (pol 7) appears to be a heterodimer comprising a catalytic subunit exhibiting 5'—)3' DNA polymerase activity and a small subunit of unknown function (Wernette and Kaguni, 1986; Insdorf and Bogenhagen, 1989a; Gray and Wong, 1992). A 3'—>5' mismatch specific exonuclease activity is associated with pol y purified from Drosophila (Kaguni and Olson, 1989), X. laevis (Insdorf and Bogenhagen, 1989b), and human mitochondria (Gray and Wong, 1992), while sequence analysis of the gene encoding the catalytic subunit from S. cerevisiae reveals that it contains a 3'—-)5' exonuclease motif (Foury, 1989; Ito and Braithwaite, 1990). The subunit structure of y polymerase appears to be conserved in 4 O Drosophila, chicken, X. laevis, and human tissues: a 125-140 kDa catalytic subunit and a 35-54 kDa small subunit (Y amaguchi et al., 1980; Wernette and Kaguni, 1986; Insdorf and Bogenhagen, 1989a; Gray and Wong, 1992). In addition, pol 7 identified in porcine liver exhibits a native molecular weight of 140 kDa (Mosbaugh, 1988), while the yeast catalytic subunit is calculated from sequence data to be 143.5 kDa (Foury, 1989). Processive for 30-90 nucleotides (Wernette et al., 1988), pol 7 does not seem well suited for continuous replication of a 15,700 to 19,500 base pair mitochondrial genome (Wolstenholme et al., 1979; Anderson et al., 1981; Bibb et al., 1981). DNA polymerase y misincorporates 1 nucleotide per 1-5 x 105 nucleotides incorporated (Kunkel and Soni, 1988; Wernette et al., 1988; Kunkel and Mosbaugh, 1989), similar to the error rates of pol e and pol 8 in the presence of PCNA (Kunkel et al., 1987; Thomas et al., 1991). Further, the rate of DNA synthesis by ‘y polymerase of 2 nucleotides per second (Wernette et al., 1988) is Similar to the rate of 4.5 nucleotides per second observed in vivo (Clayton, 1982). Thus, DNA polymerase 7 appears to be enzymatically characteristic of mitochondrial replicative DNA polymerase with regard to its rate and fidelity of DNA synthesis, but is uncharacteristic in its processivity, assuming fully processive replication of mitochondrial DNA. However, even at its moderate processivity, a high concentration of pol 7 appears to be sufficient for complete replication of the mitochondrial genome (Wernette et al., 1988) DNA polymerase accessory factors Mitochondrial DNA polymerase processivity and primer recognition factors have not yet been identified. Mitochondrial single-stranded DNA binding protein (mtSSB) is a homotetramer (13.3-15.2 kDa monomers) that binds DNA cooperatively covering an 8 to 9 nucleotide region (Van Tuyle and Pavco, 1981; Ghrir et al., 1991; Van Dyck et al., 1992; Hoke et al., 1990). Sequence comparison of mtSSB with E. coli SSB reveals 24-30% identity and 43-50% Similarity between the two, with the highest homology in the amino terminal two-thirds of the proteins (63-76% identity in some regions; Van Dyck et al., 4 l 1992; Hoke et al., 1990; Ghrir et al., 1991; Mounolou et al., 1988). This homology suggests that subunit interaction and DNA binding domains of the two SSBs are very similar, while the domain involved in SSB interaction with other proteins is not well conserved. This conservation profile is reasonable given the similar function of $833 to coat single-stranded DNA while interacting with replication proteins which are evolutionarily widely divergent. Mitochondrial DNA polymerase activity is either stimulated up to 10-fold or inhibited up to 20-fold by the presence of mtSSB depending on the purity of the DNA polymerase and the DNA substrate and reaction conditions used (Hoke et al., 1990; Mignotte et al., 1988). The inability of mitochondrial DNA polymerase to synthesize DNA in a highly processive manner is inconsistent with models of mtDNA replication. As reviewed here, we find that replicative DNA polymerases from E. coli, bacteriophage T4, and humans generally display enzymatic characteristics consistent with their roles in vivo. However, it is clear that the context in which the rate, processivity, and fidelity of DNA synthesis are measured is critical to their accurate determinations. Therefore, we propose that mitochondrial DNA polymerase catalyzes DNA synthesis with enzymatic characteristics which correlate with models of mitochondrial DNA replication in vivo, but may require additional elements to do so. CHAPTER II PROCESSIVIT Y OF MITOCHONDRIAL DNA POLYMERASE FROM DROSOPHILA EMBRYOS: EFFECTS OF REACTION CONDITIONS 42 43 INTRODUCTION Replication of the double- stranded circular mitochondrial DNA (mtDNA) genome occurs in the mitochondrial matrix and proceeds in a unidirectional and asymmetric manner (Goddard and Wolstenholme, 1980; Clayton, 1982), suggesting the possibility of continuous synthesis of both DNA strands. Indeed, current models of animal mtDNA replication, derived primarily from biochemical and electron microscopic studies of replication intermediates, favor this possibility. Because DNA polymerase ‘y (pol y) is the sole DNA polymerase found in mitochondria (Bolden et al., 1977; Bertazzoni et al., 1977; Hubscher et al., 1977), these models predict that mitochondrial DNA polymerase catalyzes continuous DNA Strand synthesis. Continuous DNA strand synthesis in both pro- and eukaryotic DNA replication generally correlates with processive synthesis (Das and Fujimura, 1979; Fisher et al., 1979; Fay et al., 1981; Hockensmith and Bambara, 1981; Pay et al., 1982; Prelich et al., 1987 ; Hurwitz et al., 1990). Whereas DNA polymerases responsible for continuous synthesis are highly processive, catalyzing the incorporation of thousands of nucleotides (nt) into the growing DNA chain in a Single binding event, those involved in discontinuous synthesis may be processive for only tens or hundreds of nucleotides. While mitochondrial DNA polymerase might be expected to catalyze highly processive DNA synthesis, it was found previously that when assayed under conditions optimized for DNA synthetic rate, Drosophila pol y is only quasi-processive, incorporating 30-90 nt per binding event (Wemett et al., 1988). The mitochondrial matrix environment is much different from that of the nucleus: the matrix contains a higher protein concentration (calculated as ~50% by weight; Srere, 1980) and higher nucleoside triphosphate levels (Bestwick et al., 1982). In addition, the matrix volume varies up to ~2-fold depending on the metabolic state of the mitochondria (Hackenbrock, 1968), resulting in fluctuating concentrations of metabolites that contribute 44 to the compartrrrental ionic strength. These alterations may be important in the regulation of the metabolic pathways within the matrix (Srere, 1980), including mtDNA replication. Interestingly, the mechanism of DNA synthesis by E. coli DNA polymerases I and III and eukaryotic DNA polymerases a and 8 was found to vary depending on reaction conditions (Bambara et al., 1978; Kwon-Shin et al., 1987; Hohn and Grosse, 1987). The sensitivity of these replicative enzymes to their assay environments coupled with the unusual mitochondrial environment suggests that mitochondrial DNA polymerase might also be influenced by the composition of the reaction solvent. We have examined the effect of various reagents on the rate and processivity of DNA synthesis by Drosophila DNA polymerase 7 on a single-stranded DNA template to address the apparent contradiction of continuous DNA synthesis catalyzed by a moderately processive enzyme. The elucidation of in vitro reaction conditions that enable mitochondrial DNA polymerase to catalyze highly processive and efficient DNA synthesis concurrently would allow correlation of enzyme function with models of mtDNA replication, as well as suggest a mechanism for its regulation. In contrast, an inability to define such conditions may implicate as yet unidentified DNA polymerase accessory factors in mtDNA replication. 45 EXPERIMENTAL PROCEDURES MATERIALS Nucleotides and nucleic acids. Unlabeled deoxy- and ribonucleoside triphosphates were purchased from P-L Biochemicals; for use at concentrations above 30 BM, ATP, GTP, and ADP solutions were adjusted to pH 7.5 with Tris base (Research Organics). [3H]dT'I‘P was purchased from ICN Biochemicals; [a-32P]dTTP was purchased from New England Nuclear. Calf thymus DNA (highly polymerized Type I) was purchased from Sigma and was activated by partial digestion with DNase I (Boehringer Mannheim) as described by Fansler and Loch (1974). Recombinant and wild type M13 viral DNAS (10,650 and 6,407 nt, respectively) were prepared by Standard laboratory methods. Synthetic oligodeoxynucleotides (15 nt) complementary to the M13 viral DNAS were synthesized in an Applied Biosystems Model 477 oligonucleotide synthesizer. Enzymes. Drosophila DNA polymerase y (Fractions IV, V and VI) was prepared as described by Wernette and Kaguni (1986). Chemicals. All solutions were prepared in water and stored at room temperature. Polyethylene glycol (8,000 MW, Sigma) was prepared as a 50% stock solution. Polyvinyl alcohol (10,000 MW, Sigma) was prepared as a 17.5% stock solution. Glycerol (Mallinckrodt) was prepared as an 80% stock solution. METHODS DNA polymerase yassay. Reaction mixtures (0.05 ml) contained 50 mM Tris-HCl (pH 8.5), 4 mM MgC12, 10 mM dithiothreitol, 30 or 120 mM KCl, 400 ug/ml bovine serum albumin, 20 BM each of dATP, dCTP, dGTP, and [3H]dTTP (1000 cpm/pmol), 10 uM (as nt) of singly-primed M13 DNA, and 0.1 to 0.2 units of Fr VI enzyme. Incubation was at 30'C for 30 rrrinutes. Specific modifications are described in the figure legends. One unit of activity is that amount that catalyzes the incorporation of l 46 nanomole of deoxyribonucleoside triphosphate into acid insoluble material in 60 minutes at 30’C using DNase I-activated calf thymus DNA as the substrate. Here, we define Standard activity as that exhibited by pol y in the presence of 120 mM KCl on singly-primed M13 DNA. Analysis of products of processive DNA synthesis by gel electrophoresis. Reactions were as above except that reaction mixtures contained 30 11M each of dATP, dCl‘P, dGTP, and 10 M of [a-32P1drrr (2 x 104 cpm/pmol), 20 M singly-primed M13 DNA, and 0.02 units of Fr VI enzyme. Incubation was at 30'C for 30 minutes unless otherwise noted. Products to be analyzed by denaturing polyacrylamide gel electrophoresis were made 1% in SDS and 10 mM in EDTA, heated for 4 minutes at 80'C and precipitated with ethanol in the presence of 5 ug of tRNA as carrier. The ethanol precipitates were resuspended in 80% formamide, and 90 mM Tris-borate. Aliquots were denatured for 2 minutes at 100'C and electrophoresed in a 6% polyacrylamide slab gel (13 x 30 x 0.15 cm) containing 7M urea in 90 mM Tris-borate (pH 8.3) and 25 mM EDTA. Alternatively, the ethanol precipitates were resuspended in 0.3 M NaOH and 20 mM EDTA, and aliquots electrophoresed in a 1.5% (agarose) slab gel ( 13 x 18 x 0.7 cm) containing 30 mM NaCl and 2 mM EDTA in 30 mM NaOH and 2 mM EDTA. Approximately equal amounts of radioactivity (~1000 cpm) were loaded in each lane. In addition, equal sample volumes were loaded on each type of gel to allow direct comparison of product size distribution. Gels were washed in distilled water for 20 rrrinutes, dried under vacuum and exposed at (-)80‘C to Kodak X-OMAT AR X-ray film using Du Pont Quanta III intensifying screens. Quantitation of the data was performed by scanning of the autoradiographs using a Bio- Image Visage 110 digital imager. The area under the peaks was determined by computer integration analysis and was normalized to the nucleotide level to correct for the uniform labeling of the DNA products. In the determination of processivity values, the length of the primer (15 nt) was subtracted from the DNA product strand lengths. 47 RESULTS Drosophila DNA polymerase y is highly processive at KCl levels suboptimal for DNA synthesis. Condensation of the mitochondrial matrix during periods of active respiration likely results in an increase in the concentration of metabolites that contribute to ionic strength (Hackenbrock, 1968). Increasing ionic Strength decreases both the activity and processivity of E. coli DNA polymerases I and III, calf thymus DNA polymerase a, and T4 phage DNA polymerase (Bambara et al., 1978; Kwon-Shin et al., 1987; Hohn and Grosse, 1987; Newport et al., 1980). Because Drosophila mitochondrial DNA polymerase is stimulated by moderate salt (Wernette et a1. 1988; Wernette and Kaguni, 1986), we investigated its processivity on singly-primed M13 DNA under conditions of varying monovalent salt concentration. Pol 7 activity varied =6-fold when assayed over the range of 0-210 mM KCl, with an optimum at ~120 mM KCl (Fig. 6A). At the same time, processivity varied dramatically (=600-fold), with the greatest abundance of full length products synthesized in the absence of KCl (Fig. 6B). Reduced polymerase activity under conditions where pol 'y is capable of copying a complete 6407 nt template in a single binding event suggests that either primer recognition or enzyme dissociation is rate limiting. To investigate the effects of other reagents on enzyme activity and processivity, subsequent assays were conducted at 120 mM KCl (moderate salt), where pol yexhibits its highest (standard) activity yet is only moderately processive (average processive unit (apu) of 45 nt) and at 30 mM KCl (low salt) where it is less active (25% of standard activity), but highly processive (apu of 2500 nt). pH has little effect on the processivity of Drosophila mitochondrial DNA polymerase. The mitochondrial inner membrane is generally impermeable to charged and highly polar molecules, allowing the formation of a pH gradient across the lipid bilayer. 48 FIGURE 6. KCl alters the rate and processivity of DNA synthesis by Drosophila pol 7. A. The rate of DNA synthesis was determined as described under "Methods" in the presence of the indicated KCl concentrations. B. Product DNA Strands were analyzed on denaturing 1.5% agarose (upper panel) and 6% polyacrylamide (lower panel) gels as described under "Methods." Adjacent lanes represent samples obtained after 20 and 40 nrinutes of incubation at 30°C and containing no KCl (lanes 1 and 2; average processive unit (apu) = 3400 nt), 30 mM KCl (lanes 3 and 4; apu = 2500 nt), 60 mM KCl (lanes 5 and 6; apu = 1600 nt), 90 mM KCl (lanes 7 and 8; apu = 140 nt), 120 mM KCl (lanes 9 and 10; apu = 45 nt), 160 mM KCl (lanes 11 and 12; apu = 20 nt), 210 mM KCl (lanes 13 and 14; apu = 6 nt). Numbers on the left of the photograph indicate the position and size (in nt) of Hpa H restriction fragments of M13Goril replicative form DNA (Kaguni and Ray, 1979) and Hind III restriction fragments of lambda DNA that were electrophoresed in adjacent lanes. In the determination of processivity values, the length of the primer ( 15 nt) was subtracted from the DNA product strand lengths measured relative to the molecular weight markers shown. Products obtained after 40 minutes of incubation were similar in size and distribution to those obtained after 20 minutes of incubation, indicating that they result from single binding events; subsequent analyses were performed using 30 minute incubations at 30°C. '49 0 50 100 150 KCl, InM POLYMERASE ACTIVITY, % STD a e B 1234567891011121314 6557 _ 4 r— 195— 1207— 564: -- f 357—- .‘JA . 272— - » '1 w...,‘.. 176— , o...“ 125— "” ‘ “qt. w— I l l. 35— a” 7 '4’"! 17—. . .. ".‘r Figure 6 200 250 50 The extent of this gradient varies with matrix pH, as the cytosol seems to be effectively buffered (Moroney et al., 1984). Lowering the pH of the reaction solvent from pH 8.0 to 6.0 increased the processivities of calf thymus DNA polymerases a and 8 approximately 30-fold (Sabatino et al., 1988). In contrast, polymerase activity was reduced 5- to 7-fold at pH 6.0 relative to its optimum at pH 7.0. When assayed over the range of pH 6-10, the activity of Drosophila pol yvaried ==33.5-fold at moderate salt and =10-fold at low salt, with optimal activity achieved between pH 8.0 to 9.6 and at pH 10, respectively (Fig. 7A). However, processivity varied less than 4-fold regardless of KCl concentration, and the enzyme was most highly processive at pH 8.5 at both moderate and low salt (Fig. 7B). Thus, optimal polymerase activity and processivity were achieved concurrently at 120 mM KCl. However, like pols a and 8, pol 7 was most processive at pH suboptimal for activity at 30 mM KCl. DNA synthetic product length is diminished with increasing MgClz concentrations. DNA polymerases require magnesium as a cofactor for nucleotide incorporation in the form of a Mg-dNTP complex. Increasing MgClz concentrations decreased the processivity of calf thymus pols a and 8 while increasing their activity ' (Sabatino et al., 1988). Likewise, when assayed from 0.01-64 mM MgC12, the addition of Mg2+ decreased the processivity of pol y~6—fold at moderate salt and =700-fold at low salt, at concentrations as low as 1 mM MgC12 (Fig. 8B). Polymerase activity varies ~10-fold at 120 mM KCl and is optimal from 0.5-6 mM MgC12 (Fig. 8A). At 30 mM KCl, pol 7 activity varies =30-fold and the enzyme exhibits two optima at 0.25 and 20-25 mM MgC12. Thus, the enzyme is capable of synthesizing long products (>3000 apu) at a high rate (0.2- 0.5 mM MgC12 at 30 mM KCl), yet is also able to make short products (5 45 apu) quickly (0.25-7 mM MgClz at 120 mM KCl, and 18-27 mM MgC12 at 30 mM KCl). 51 FIGURE 7. pH affects the rate and processivity of DNA synthesis by Drosophila pol 7. A. The rate of DNA synthesis was determined as described under "Methods" in the presence of 50 mM TriS°HCl (pH 6-9.6) or 50 mM CAPS -KOH (pH 9.4-12) and 30 mM (open circles) or 120 mM (closed circles) KCl. B. Product DNA strands were analyzed on denaturing 1.5% agarose (upper panel) and 6% polyacrylamide (lower panel) gels. Reactions were performed at 30 mM KCl (lanes 1-3) or 120 mM KCl (lanes 4-6) and pH 7.0 (lanes 1 and 4; apu = 780 nt and 35 nt respectively), pH 8.5 (lanes 2 and 5; apu = 2800 nt and 37 nt), and pH 10.0 (lanes 3 and 6; apu = 1200 nt and 38 nt). 52 8.0 9.0 10.0 11.0 7.0 A 0 i.<4ru<.4.< - 6 120 00 0 0 0 0 0 :hm 8 .>.=>_.—.U< umdOm B “123 456 6557 — 4361 — 1925 —- 1207 muz: 357 — 272 — 176 — 125 — 35— Figure 7 53 FIGURE 8. MgClz alters the rate and processivity of DNA synthesis by Drosophila pol y. A. The rate of DNA synthesis was determined as described under "Methods" in the presence of the indicated MgClz concentrations and 30 mM (open circles) or 120 mM (closed circles) KCl. B. Product DNA strands were analyzed on denaturing 1.5% agarose (upper panel) and 6% polyacrylamide (lower panel) gels. Reactions were performed at 30 mM KCl (lanes 1-9) or 120 mM KCl (lanes 10-16) and 0.15 mM MgC12 (lanes 1 and 10; apu = 4300 nt and 40 nt respectively), 0.3 mM MgC12 (lanes 2 and 11; apu = 4300 nt and 40 nt), 0.5 mM MgC12 (lanes 3 and 12; apu = 3000 nt and 40 nt), 1 mM MgC12 (lanes 4 and 13; apu = 2900 nt and 25 nt), 4 mM MgClg (lanes 5 and 14; apu = 2100 nt and 25 nt), 10 mM MgC12 (lanes 6 and 15; apu = 25 nt and 15 nt), 20 mM MgClg (lanes 7 and 16; apu = 10 nt and 7 nt), 30 mM MgC12 (lane 8; apu = 6 nt), 40 mM MgC12 (lane 9; apu = 6 nt). 54 A 12°" :3; ”- 1001 W . 40' 80 2M v . , . t 0.0 0.5 1.0 15 1.0 POLYMERASE ACTIVITY, ‘70 STD S 0 20 40 MgClz B 123456789 1111121314516 6557 — ' 4361 — m a ‘1 1925 — '.' 12m _ 564 : - 357 — m _ ' m _ ' ”" ~- 125 — l .- 00 ”Q... ‘0 __ 35 — ‘ 17 — ‘ . Figure 8 60 80 55 _ ATP specifically stimulates DNA synthesis by Drosophila'ypolymerase. Seventy percent of the total cellular ATP is localized within the mitochondrial matrix (Hutson et al., 1989), making it at least 4-fold more abundant than the other. nucleoside triphosphates present (Bestwick et al., 1982). ATP hydrolysis is involved in primer recognition by, and increases the processivities of prokaryotic, viral and eukaryotic DNA polymerases (Burgers and Kornberg, 1982b; Cha and Alberts, 1988; Tsurimoto and Stillman, 1990; Burgers, 1991). ATP, but not GTP or ADP, stimulated the activity of Drosophila mitochondrial DNA polymerase 4-fold at low salt in the presence of ATP concentrations from 7.5 to 12 mM, such that the activity observed at moderate salt in the absence of ATP was achieved (Fig. 9A, B, C). This specific stimulation by ATP was accompanied by a non-proportional decrease in processivity (Fig. 9D). Notably, while ATP did not stimulate activity at 120 mM KCl, it decreased processivity above 5 mM ATP (Fig. 9D), as did GTP and ADP at both moderate and low KCl concentrations (data not shown). Titration experiments indicate that chelation of Mg2+ by ATP is unrelated to the Specific stimulation observed: the MgClz optimum remains at 4 mM as the ATP concentration is varied from 5 to 12 mM (data not Shown). Effects of macromolecular crowding agents. Protein concentration in the mitochondrial matrix has been calculated to be =50% by weight; thus, the behavior of matrix enzymes may be expected to lie between that of enzymes in solution and that of enzymes in ordered complexes (Srere, 1980). The conditions provided by macromolecular crowding agents in vitro may mimic those found in the matrix. Crowding agents (dextran and polyethylene glycol (PEG)) enhance binding of E. coli DNA polymerase I and T4 DNA polymerase to template-primers, resulting in increased activity, yet having no effect on processivity (Zimmerman and Harrison, 1987; Jarvis et al., 1990). Protein-protein interactions are also enhanced, as indicated by an increased processivity of T4 DNA polymerase in the presence of its accessory proteins (Jarvis et al., 1990). Drosophila ‘y 56 FIGURE 9. Efi'ect of ATP, GTP, and ADP on the rate and ATP on the processivity of DNA synthesis by Qmsgphfla pol 7. The rate of DNA synthesis was determined as described under "Methods" in the presence of the indicated concentrations of ATP (A), GTP (B), or ADP (C) and 30 mM (open circles) or 120 mM (closed circles) KCl. D. Product DNA strands were analyzed on denaturing 1.5% agarose (upper panel) and 6% polyacrylamide (lower panel) gels. Reactions were performed at 30 mM KCl (lanes 1-6) or 120 mM KCl (lanes 7-11) and no ATP (lanes 1 and 7; apu = 3000 at and 95 nt respectiveIY). 2 mM ATP (lanes 2 and 8; apu = 2900 nt and 80 nt), 5 mM ATP (lanes 3 and 9; apu = 3300 nt and 80 nt), 10 mM ATP (lanes 4 and 10; apu = 1800 nt and 50 nt), 15 mM ATP (lanes 5 and 11; apu = 350 nt and 40 nt), 25 mM ATP (lane 6; apu = 80 nt). 57 7 891011 17— 1‘ .‘ .403 I ' M 1H. D _ _ _ _ __ __ _ _ _ _ mm H m a mu m u w B 0 3 .5 2 v .0 2 0 .2 .HM m. 0 .WA .m 0 .5 A B . 1 . . . S 0 I . . . «(a 0 mmmmwmo Mwmwmmma ‘- 1 1 1 95 a» .>._.—>_._.0< Hmdg 9.5 cm .>.=>=.U< mw<¢m2>40m GTP, mM .3 r .HM m m. .mA -5 C . .1 . . . S 0 m m w w w m o l 1 Eu .8 .>._._>:b< magma—as?— Figure 9 58 polymerase exhibited a 7-fold variation in activity upon the addition of PEG in the range of 0%-24%: activity decreased in the range of 4% to 12% at both 120 and 30 mM KCl, and was restored either partially or fully at ==.16% PEG at moderate and low salt, respectively (Fig. 10A). pol 7 synthesized longer DNA products in the presence of 8% PEG at both 120 mM and 30 mM KCl (Fig. 103), where its activity was 2- to 3-fold reduced. In contrast, polyvinyl alcohol has little effect on either the activity or processivity of the mitochondrial DNA polymerase (data not shown). Glycerol stimulates DNA polymerase activity and processivity concurrently. Glycerol acts to stabilize enzyme activity and conformation (Gekko and Timasheff, 1981), and may enhance the association of proteins in solution. Drosophila pol 7 activity was stimulated approximately 2-fold when assayed in the presence of ~20% glycerol at both moderate and low salt (Fig. 11A). In addition, its processivity increased 1.5- to 3.5-fold with the inclusion of =15% glycerol (Fig. 11B), demonstrating that pol yis capable of exhibiting optimal activity and processivity concurrently at each KCl level examined in the presence of glycerol. Limited template-primer availability does not alter the processivity of Drosophila ypolymerase. Protein-protein interactions between DNA polymerase molecules and/or accessory proteins have been demonstrated in both prokaryotic and eukaryotic systems (Kornberg and Baker, 1992). To promote such interactions between pol 7 molecules and/or putative sub-stoichiometric accessory proteins, we analyzed enzyme activity under conditions of limiting primer termini. After preincubation of the near- homogeneous enzyme with an M13 DNA template containing 5'-end labeled primers, DNA synthesis was carried out in the presence of a 45-fold excess of unlabeled DNase I- activated calf thymus DNA which serves to trap unbound enzyme molecules, thereby ensuring a single DNA synthetic cycle. The processivity of Drosophila y polymerase was 59 FIGURE 10. PEG alters the rate and processivity of DNA synthesis by Drosophila pol ‘y. A. The rate of DNA synthesis was determined as described under "Methods" in the presence of the indicated PEG concentrations and 30 mM (open circles) or 120 mM (closed circles) KCl. B. Product DNA strands were analyzed on denaturing 1.5% agarose (upper panel) and 6% polyacrylamide (lower panel) gels. Reactions were performed at 30 mM KCl (lanes 1-5) or 120 mM KCl (lanes 6-10) and no PEG (lanes 1 and 6; apu = 2500 nt and 45 nt respectiveIY). 4% PEG (lanes 2 and 7; apu = 4000 nt and 50 nt), 8% PEG (lanes 3 and 8; apu = 4100 nt and 330 nt), 14% PEG (lanes 4 and 9; apu = 4000 nt and 100 nt), 18% PEG (lanes 5 and 10; apu = 2400 nt and 40 nt). l :- § 3383 O 0 10 POLYMERASE ACTIVITY, ‘70 STD PEG, % B 12345 678910 6557 — ’ 4361 —— 1925 — 1207 — 564'— 357 — '5 , 272 — '1 176 _ 125 —- 35_ "to 0 Figure 10 61 FIGURE 11. Gycerol increases the rate and processivity of DNA synthesis by Wild pol y. A. The rate of DNA synthesis was determined as described under "Methods" in the presence of the indicated glycerol concentrations and 30 mM (open circles) or 120 mM (closed circles) KCl. B. Product DNA strands were analyzed on denaturing 1.5% agarose (upper panel) and 6% polyacrylamide (lower panel) gels. Reactions were performed at 30 mM KCl (lanes 1-5) or 120 mM KCl (lanes 6-10) and no glycerol (lanes 1 and 6; apu = 2900 nt and 45 nt respectiveIY). 7.5% glycerol (lanes 2 and 7; apu = 3600 nt and 85 nt), 15% glycerol (lanes 3 and 8; apu = 4400 nt and 140 nt), 30 % glycerol (lanes 4 and 9; apu = 4200 nt and 155 nt), 45% glycerol (lanes 5 and 10; apu = 2400 nt and 170 nt). 62 50. (W POLYMERASE ACTIVITY, % STD 0 10 20 30 40 GLYCEROL. % B 12345 678910 6557 _ 4361 — 1925 — 'l- i S ,7 ”:3. 1207 — ‘ ’ 'r . 564 : , , s . . '9‘ 357 _ . 1 . o- 272 — 000.‘ 176 — O... 125 — “00' 60 _ 35 — fi ' .24.: Figure 11 60 63 unaffected when the template-primer to enzyme molecule ratio was varied over a 60-fold range, from 0.5 to 30 primer termini per pol 7 molecule (Fig. 12). FIGURE 12. Varying the p01 7 .‘ template-primer molecular ratio has little effect on the processivity of DNA synthesis by Drosophila pol 7. DNA synthesis reactions were carried out in the presence of a DNA “trap” to ensure processive DNA synthesis. Reaction mixtures were as described under "Methods," except that they contained 2 M, 8 M, or 30 11M 5’-end labeled singly-primed M13 DNA and 0.4, 0.2, or 0.1 units of pol 7, respectively. After incubation at 30‘C for 5 minutes, prewarmed (30°C) DNase I -activated calf thymus DNA (360 11M) and 30 11M each of dATP, dCTP, dGTP, and dTTP were added simultaneously. Reactions were incubated further at 30'C for 30 nrinutes, and the product DNA strands isolated and analyzed on denaturing 1.5% agarose (upper panel) and 6% polyacrylanride (lower panel) gels. Reactions were performed at 30 mM KCl (lanes 1- 3) or 120 mM KCl (lanes 4-6) at pol y to template-primer ratios of 2:1 (lanes 1 and 4), 1:4 (lanes 2 and 5), 1:30 (lanes 3 and 6). 65 1 2 3 4 S 6 6557 — .0 4361 — ' in 1925 — 1207 _ 564 : ‘ 357 - E 272 — p‘ *0. 176 — ... -o 125 -— ~ 2 60 _ 35 — 17— A A... Figure 12 66 DISCUSSION Replication of the Drosophila mitochondrial genome proceeds asynchronously by a mechanism in which up to 98% of the leading DNA strand is copied prior to initiation of lagging DNA strand synthesis (Goddard and Wolstenbolme, 1978; Wolstenholme et al., 1979). Although continuous DNA strand synthesis would be consistent with this mechanism, biochemical characterization of Drosophila mitochondrial DNA polymerase does not fully support it; DNA polymerases implicated in continuous DNA strand synthesis are generally highly processive, yet it was previously shown that pol y is only moderately processive under reaction conditions optimal for DNA synthetic rate (Wernette et al., - 1988). By altering various parameters of in vitro DNA synthesis, we hoped to increase the processivity of y polymerase while maintaining a high rate of nucleotide polymerization. We Show that mitochondrial DNA polymerase, like other pro- and eukaryotic DNA polymerases, is sensitive to its assay environment, but that changes in reaction conditions yield a highly processive enzyme only under conditions that are suboptimal for DNA synthetic rate. As incorporation of a nucleotide is generally more rapid than dissociation and reassociation of a DNA polymerase at the primer terminus (Kornberg and Baker, 1992), we might expect pol y to be most active under conditions that limit enzyme cycling, that is, under conditions favoring high processivity. Instead, our results suggest that initiation and/or termination of a processive product are even more rate limiting. Increasing concentrations of KCl induced a 6-fold increase in the activity, but a 600-fold diminution in the processivity of Drosophila pol ‘y. Ionic strength affects DNA and protein structure as well as molecular interactions, and thus it likely affects the binding of 'ypolymerase to DNA. Because high processivity correlates with strong enzyme-primer terminus interactions, our data suggests that increasing ionic strength destabilizes them. This destabilization may allow rapid enzyme cycling, resulting in elevated activity if cycling is the rate limiting step. In addition, the stabilization of DNA secondary Structure at high ionic strength (Lyons and Kotin, 1964) likely contributes to the multitude of pause sites 67 observed under these conditions. In contrast, at low ionic strength, the DNA polymerase is tightly bound to the primer terminus and meets fewer impediments on the template DNA, facilitating fully processive DNA synthesis. Here, strong enzyme-DNA associations may inhibit enzyme cycling, resulting in limited overall activity. Alterations in pH resulted in differential activity optima for Drosophila pol ‘y at low versus moderate salt. However, the enzyme was most processive at pH 8.5 regardless of the KCl level. The different activity optima likely reflect ionic strength effects on shielding of amino acid side groups. At moderate salt, it appears that side groups which are titratable in the range of pH 8.0 to pH 9.6 are sufficiently shielded such that the changes induced lack significant effects on the structure, and therefore the function, of pol 7. On the other hand, at low salt, titration of Side groups at more basic pH results in increased DNA polymerase activity. Here, the intrinsic charges of incompatible amino acid side chains may be altered sufficiently to allow their association, stabilizing enzyme structure and stimulating activity. M gC12 decreased the processivity of the Drosophila mitochondrial DNA polymerase, yet the optimal Mg2+ levels for polymerase activity varied at low versus moderate salt. Mg2+ is bound by both DNA and DNA polymerases, and is a required cofactor for enzyme activity, as Mg-dNTP complexes are the substrates for nucleotide polymerization. MgClz also contributes to the ionic strength of the reaction solvent, thus partially explaining the increase in activity at high MgC12 levels at low salt, yet the loss of polymerase activity with the addition of MgC12 at moderate salt. However, this “salt” effect is probably not responsible for the initial peak in DNA polymerase activity observed between 0.25 and 0.5 mM MgC12 at low salt. Further, although differential binding of Mg2+ by the DNA, dNTPs and pol y may result in a rapid initial increase in the rate of DNA synthesis, the presence of an approximately 20-fold excess of Mg2+ molecules over potential binding sites renders this potential explanation unlikely as well. Interestingly, E. coli DNA polymerase I possesses at least three types of divalent cation binding sites 68 exhibiting varying affinities for manganese: the high and intermediate (3-10 pM) affinity sites appear to be stimulatory, while the low affinity sites (800-900 11M) seem to be inhibitory (Slater et al., 1972). Such a binding scenario involving 7 polymerase could explain the observed initial peak of polymerase activity. At the same time, diminished processivity may result from the increase in ionic strength due to addition of MgC12_ Alternatively, the decrease may be related to the finding of Griep and McHenry (1988) that the inclusion of Mg2+ alters the conformation of the processivity factor of E. coli DNA polymerase III, the 37 kDa B subunit: the presence of 10 mM MgClg specifically Shifts the B subunit from a dimer into a predominantly monomeric form. While the dimeric form is directly implicated in processive DNA synthesis by pol III holoenzyme (Burgers and Kornberg, 1982b; Johanson and McHenry, 1980; Kong et al., 1992), the role of the monomer has not been addressed. Drosophila mitochondrial DNA polymerase comprises a 125 kDa polymerase catalytic subunit and a 35 kDa subunit of unknown function (Wernette and Kaguni, 1986). A pol III B subunit-like activity which is affected Similarly by Mg2+ may be present in the two subunit pol 7. High levels of ATP, but not GTP or ADP, were able to restore polymerase activity when Drosophila pol 7 was assayed at low salt. .The Specificity of this stimulation eliminates the possibility that the ionic strength contribution of added nucleotides alone is responsible. Nucleotide hydrolysis may be involved, as there is some stimulation of pol 7 activity by GTP but no effect of ADP. However, we were unable to detect any DNA- dependent ATPase activity in Drosophila pol 7 (CM. Wernette, A.J. VonTom, and LS. Kaguni, unpublished observations). Lack of ATP hydrolysis may be indicative of the lack of a protein factor or factors which work(s) to chaperone the putative processivity factor onto the DNA (Tsurimoto and Stillman, 1990; O'Donnell, 1987; Maki and Kornberg, 1988). Alternatively, ATP bound to pol 7 may induce a conformational change that influences polymerase catalytic efficiency. It is clear, although perhaps surprising, that stimulation by ATP is not a result of lowering the effective concentration of Mg“ via 69 chelation; when the ATP concentration is varied from 5 to 12 mM, the MgC12 optimum remains at 4 mM (A.J. VonTom, unpublished observation). In addition, GTP would be expected to chelate the Mg2+ as well as ATP, yet its effect on enzyme activity is minimal. The macromolecular crowding agent, polyethylene glycol, stimulated DNA polymerase activity when assayed at low salt, yet inhibited and then partially restored activity when assayed at moderate salt. Enhanced processivity was observed at both KCl levels, but was not coincident with optimal activity. These results may be complicated by the ability of PEG to precipitate protein, DNA, and protein-DNA complexes. However, PEG was shown to enhance the association of E. coli DNA polymerase I, its Klenow fragment, and phage T4 DNA polymerase with DNA (Zimmerman and Harrison, 1987), resulting in increased DNA polymerase activity. Stimulation was generally most pronounced at high ionic strength, such that the optimal salt concentration for enzyme activity increased with increasing PEG. Predictably, there was no apparent effect on DNA polymerase processivity since protein-DNA association, but not dissociation should be affected by crowding (Minton, 1983). In contrast, PEG enhanced the assembly of a holoenzyme form of phage T4 DNA polymerase and consequently, its processivity (Jarvis et al., 1990). Our data with pol y are consistent with either possibility: PEG may enhance binding of y polymerase to the DNA as a consequence of macromolecular crowding, and/or may promote protein-protein interactions (either polymerase-polymerase or polymerase- accessory factor). Glycerol may also promote these interactions, because its inclusion increased both DNA polymerase activity and processivity, regardless of salt concentration. The processivity of Drosophila pol 1 was unchanged when the enzyme molecule to primer terminus ratio was varied over a 60-fold range, perhaps reflecting an inability to enhance protein-protein interactions by non-chemical means in the near-homogeneous enzyme. Alternatively, some associations which occur may not be detectable because they do not alter the processivity of mitochondrial DNA polymerase. 70 We have shown here that Drosophila ‘y polymerase is capable of fully processive DNA synthesis on a natural DNA template in vitro only under conditions that are suboptimal for DNA polymerase activity. Thus, it is difficult to make a direct correlation of pol '7 function with current models of mitochondrial DNA replication or to suggest a mechanism for its regulation. The ATP/ADP ratio and the pH of the mitochondrial matrix have been implicated in the control of oxidative phosphorylation (Brand and Murphy, 1987). Further, the ATP/ADP ratio was shown to be the primary regulator of the tricarboxylate cycle in insect flight muscle (I-lansford, 1980). The mechanism of control of DNA replication in the mitochondrion is unknown, but the ability of mitochondrial DNA polymerase to catalyze DNA synthesis under a wide range of experimental conditions suggests that replication can occur in actively respiring, as well as resting organelles. In contrast, only a narrow set of conditions facilitate processive DNA synthesis, possibly restricting continuous DNA synthesis to Specific periods of mitochondrial activity. Although the aqueous in vitro assay environment may be too unlike that of the mitochondrial matrix to draw accurate conclusions regarding the mechanism of pol 7 function, our data suggests that other protein factors involved in mitochondrial DNA replication may function to promote high processivity under conditions optimal for DNA synthetic rate. These protein factors may associate with pol y or, as suggested by Sabatino et al. (1988), they may exert their effects by altering the microenvironment to which the DNA polymerase is exposed. Furthermore, using prokaryotic, viral, and eukaryotic nuclear systems as precedent, we would predict that mitochondrial DNA polymerase associates with several accessory proteins involved in primer recognition, processivity enhancement, and Single-stranded DNA coating. We hope to gain insight into the function of such accessory proteins in mitochondrial DNA replication by reinvestigating the effects of the various reaction parameters examined here, on pol y in association with such factors, as they are identified. CHAPTER III ESCHERICHIA COLI SINGLE-STRANDED DNA BINDING PROTEIN STIMULATES MIT OCHONDRIAL DNA POLYMERASE FROM DROSOPHILA EMBRYOS 71 72 INTRODUCTION Single-stranded DNA binding protein (SSB) from E. coli has numerous roles in the replication of the bacterial genome. In addition to protecting sin gle-stranded DNA from nuclease digestion (Meyer and Laine, 1990), it enhances helix destabilization by DNA helicase by preventing renaturation of unwound duplex (Baker et al., 1987), and helps organize and stabilize replication origins (Baker etal., 1986). Further, SSB enhances the fidelity of DNA polymerase, likely by increasing the rigidity of the template DNA, promoting increased steric hindrance between template and incorrect nucleotides (Kunkel et al., 1979; 1983). Finally, SSB increases the processivity of DNA polymerase by destabilizing the DNA secondary structure which normally causes polymerase pausing and dissociation (LaDuca et al., 1983). In fact, the presence of E. coli SSB in sufficient amounts to completely coat all of the ssDNA present in a reaction results in stimulation of E. coli DNA polymerase II and III holoenzyme (Sigal et al., 1972; McHenry, 1988), bacteriophage T7 DNA polymerase (Tabor et al., 1987)), herpes simplex virus-1 DNA polymerase (O'Donnell et al., 1987), and human DNA polymerase 8 holoenzyme (Kenny et al., 1987). In contrast, SSB has no effect on E. coli DNA polymerase I (Molineux et al., 1974) or bacteriophage T4 DNA polymerase (Fay et al., 1982). The mitochondrial analog to SSB (mtSSB) has been identified and purified from Saccharomyces cerevisiae (Jong et al., 1985), Xenopus laevis (Mignotte et al., 1985), and rat tissues (Pavco and Van Tuyle, 1985). The gene encoding the S. cerevisiae (Van Dyck et al., 1992), and the cDNAs encoding the X. laevis (T iranti et al., 1991), rat (Tiranti et al., 1993) and human (Tiranti et al., 1993) mtSSBs have been cloned and sequenced. Mitochondrial SSB is a 13.3-15.3 kDa monomer with a native molecular weight of 56.2 kDa, implying that it is a tetramer in solution (Pavco and Van Tuyle, 1985; Ghrir et al., 1991; Van Dyck et al., 1992; Tiranti et al., 1993). It binds DNA cooperatively with a 73 binding site size of 8-9 nucleotides and has an affinity for DNA that is similar to that displayed by E. coli SSB (Mignotte, et al., 1985; Hoke et al., 1990). Mitochondrial SSB is required for mitochondrial DNA (mtDNA) maintenance in yeast, in which it was identified as a suppressor of mitochondrial thermosensitivity associated with a disruption of a mtDNA helicase gene (Van Dyck et al., 1992). Its role in preventing the renaturation of displacement loops in vertebrate mtDNA replication likely aids in enhancing helicase activity (Van Tuyle and Pavco, 1981; Pavco and Van Tuyle, 1985). Further, mtSSB has been shown to coat the displaced ssDNA that is the template for lagging strand synthesis in mtDNA replication (Pavco and Van Tuyle, 1985), and is proposed to aid in the prevention of non-specific lagging DNA strand initiation by mtDNA primase (Wong and Clayton, 1985; Ledwith et al., 1986). The effect of mtSSB on mitochondrial DNA polymerase (pol y) is unclear. While rat mtSSB increased the activities of both pol y and E. coli DNA polymerase I =10-fold on poly(dT)-oligo(dA), a synthetic homo-polymer of low primer density, it had no effect on pol 7 on poly(dA)°oligo(dT) for which it exhibits low affinity (Hoke et al., 1990). In contrast, X. laevis mtSSB had no effect on DNA synthesis in a mitochondrial lysate, but stimulated partially purified pol y ==v3-fold on a template with high primer density, poly(rA)-oligo(dT), and ~l.5-fold on poly(dA)-oligo(dT), while having no effect on either E. coli DNA polymerase I or rat DNA polymerase 0t (Mignotte et al., 1988). However, with the use of singly-primed sin gle-stranded viral DNA as template, mtS SB was completely inhibitory to mitochondrial DNA polymerase activity (Mignotte et al., 1988). Comparison of the yeast mtSSB amino acid sequence with that of E. coli SSB indicated that the two were 24% identical and 50% similar (Van Dyck et al., 1992). This homology was to the amino-terminal two-thirds of E. coli SSB which encompasses its DNA binding domain (Meyer and Laine, 1990; Van Dyck et al., 1992). In fact, three anrino acids which are known to be involved in DNA binding by E. coli SSB are well conserved in the mtSSBs: tryptophan at position 54 and phenylalanine at position 60 are 74 identical in all of the mtSSBs identified, while tryptophan at position 40 is identical in human, rat, and Xenopus SSBS, but not in yeast where an arginine is found instead (Van Dyck et al., 1992; Tiranti et al., 1993). Phenylalanine 60 is the site of DNA crosslinking to E. coli SSB (Merrill et al., 1984) and its role in DNA binding, as well as those of tryptophan 40 and 54, was confirmed by site directed mutagenesis (Khamis et al., 1987; Casas-Finet et al., 1987). The amino acid sequence of rat mtSSB is 30% identical and 50% similar overall to E. coli SSB, while that of human is 32% identical and 50% similar (T iranti et al., 1993). Interestingly, human mtSSB is more similar to E. coli SSB than to yeast mtSSB (18% identical and 38% similar) even though it is highly homologous to both rat (91% identical) and Xenopus (79% identical) mitochondrial SSBs (Tiranti et al., 1993). Mitochondrial DNA polymerase is capable of highly processive DNA synthesis only under conditions that are subOptimal for DNA synthetic rate. While mitochondrial DNA replication in vivo is slow (approximately 4.5 nucleotides per second per DNA strand; Clayton, 1982), DNA polymerase catalyzes DNA synthesis at an even lower rate when carrying out highly processive DNA synthesis in vitro (approximately 0.5 nucleotides per second per DNA strand; Wernette et al., 1988 and this report). Because mitochondrial SSB is critical for mtDNA replication in vivo and appears to affect the activity of pol y, we have examined the effect of E. coli SSB on mitochondrial DNA polymerase from D. melanogaster. Although Drosophila mtSSB has not yet been identified, the high amino acid sequence conservation among mtSSBs and with E. coli SSB suggests that its effects on y polymerase may reflect that of mtSSB as well. 75 EXPERIMENTAL PROCEDURES MATERIALS Nucleotides and nucleic acids. Unlabeled deoxy- and ribonucleoside triphosphates were purchased from P-L Biochemicals. [3H]dT'l‘P was purchased from ICN Biochemicals; [or-32P]dTI‘P and [y-3ZP]ATP was purchased from New England Nuclear. Recombinant and wild type M13 viral DNAS (10,650 and 6,407 nt, respectively) were prepared by standard laboratory methods. Synthetic oligodeoxynucleotides (15 nt) complementary to the M13 viral DNAS were synthesized in an Applied Biosystems Model 477 oligonucleotide synthesizer. Enzymes. Drosophila DNA polymerase 7 (Fraction VI) was prepared as described by Wernette and Kaguni (1986). Drosophila SSB (RF-A; DSSB) was generously provided by Dr. LR. Lehman of Stanford University. E. coli SSB and bacteriophage T4 gp32 were from United States Biochemical. METHODS DNA polymerase yassay. Reaction mixtures (0.05 ml) contained 50 mM TrissHCl (pH 8.5), 4 mM MgC12, 10 mM dithiothreitol, 30 or 120 mM KCl, 400 ug/ml bovine serum albumin, 20 11M each of dATP, dCTP, dGTP, and [3H]dT'TP (1000 cpm/pmol), 10 M (as nt) of singly-primed recombinant M13 DNA, and 0.1 to 0.2 units of Fr VI enzyme (3- to 6-fold excess of primer ends over pol 7 molecules). Incubation was at 30'C for 30 nrinutes. Specific modifications are described in the figure legends. One unit of activity is that amount that catalyzes the incorporation of 1 nanomole of deoxyribonucleoside triphosphate into acid insoluble material in 60 nrinutes at 30'C using DNase I-activated calf thymus DNA as the substrate. Here, we define standard activity as that activity exhibited by pol y in the presence of 120 mM KCl on singly primed M13 DNA. 76 Analysis of products of processive DNA synthesis by gel electrophoresis. Reactions were as above except that reaction mixtures contained 30 11M each of dATP, dCTP, dGTP, and 10 11M of [or-32P]dT'I'P (2 x 104 chpmol), 20 11M singly-primed wild type M13 DNA, and 0.02 units of Fr VI enzyme (60-fold excess of primer ends over pol 7 molecules). Incubation was at 30'C for 4 or 8 minutes. Products were made 1% in SDS and 10 mM in EDTA, heated for 4 minutes at 80'C, phenol-chloroform extracted, and precipitated with ethanol in the presence of 0.5 ug of tRNA as carrier. The ethanol precipitates were resuspended in 30 mM NaOH and 20 mM EDTA, and electrophoresed in . a 1.5% (agarose) slab gel (13 x 18 x 0.7 cm) containing 30 mM NaCl and 2 mM EDTA in. 30 mM NaOH and 2 mM EDTA. Approximately equal amounts of radioactivity (~1000 cpm) were loaded in each lane. The gel was washed in distilled water for 20 minutes, dried under vacuum and exposed at (-)80'C to Kodak X-OMAT AR X-ray film using a Du Pont Quanta III intensifying screen. Time course of pol 7 DNA synthesis. Reaction mixtures were as for the DNA polymerase assay, except were 450 [.11. contained 0.9 units of Fraction VI enzyme (6-fold excess of primer ends over pol 7 molecules), and dNTPs (1000 cpm/pmol or 3000 cpm/pmol) were initially omitted. Incubation was for 5 minutes at 30'C, dNTPs were added, and additional incubation was as indicated in the figure legends. Aliquots (0.05 ml) were stopped by addition to 1 ml of 10% TCA and 10 mM sodium pyrophosphate. For analysis of products by gel electrophoresis, 32P-dT'l‘P was included in place of 3H-dTTP. Aliquots were processed and electrophoresed as for the processivity assay. Primer extension assay. Reaction mixtures (0.05 ml) contained 50 mM Tris-HCl (pH 8.5), 4 mM MgC12, 10 mM dithiothreitol, 30 mM KCl, 400 ttg/ml bovine serum albumin, 30 BM each of dATP, dGTP, and dTTP, 20 11M (as nt) of 5'-end labelled singly- primed M13 DNA, and 0.2 units of Fraction VI enzyme (3-fold excess of primer ends over pol 7 molecules). Incubation was at 30'C for 5 minutes. Samples were made 1% in SDS and 10 mM in EDTA, heated for 10 minutes at 65'C, phenol-chloroform extracted, and 77 precipitated with ethanol in the presence of 0.5 pg of tRNA as carrier. The ethanol precipitates were resuspended in 80% formamide and 90 mM Tris borate. Aliquots were denatured for 2 minutes at 100'C and electrophoresed in an 18% polyacrylamide slab gel (13 x 24 x 0.075 cm) containing 7 M urea in 90 mM Tris-borate (pH 8.3) and 25 mM EDTA. 78 RESULTS E. coli SSB stimulates DNA polymerase yactivity. E. coli SSB was previously shown to have no effect on the activity of partially purified X. laevis mitochondrial DNA polymerase (Mignotte et al., 198 8). However, when nearly homogeneous D. melanogaster 'y polymerase was assayed in the presence of SSB and 120 'mM KCl, we found that polymerase activity was stimulated approximately 2- to 3-fold (200% to 300% of standard activity; Figure 13). Further, when pol 7 was assayed in the presence of SSB and 30 mM KCl, activity was stimulated approximately 25-fold (625% of standard activity), close to a level achieved only on DNase-activated calf thymus DNA optimized for y polymerase activity. Maximal stimulation was observed when the amount of SSB present was sufficient to coat all of the single-stranded DNA. Because the presence of salt alters the binding characteristics of E. coli SSB, the single-Stranded DNA is saturated with binding protein at 0.5 ug and 1.0 ug of SSB in the presence of 120 mM and 30 mM KCl, respectively (Lohman and Overman, 1985). We had found previously that the mechanism of DNA synthesis by pol 7 was dependent on the KCl concentration in which it was assayed: At 120 mM KCl, 7 polymerase was most active (standard activity) yet only moderately processive (average processive unit of 45 nucleotides), while at 30 mM KCl, 7 polymerase was less active (25% of standard) but highly processive (average processive unit of 2500 nucleotides). Therefore, we investigated the effect of KCl concentration on the stimulation of pol y by E. coli SSB. Stimulation of mitochondrial DNA polymerase by SSB was maximal (25-fold) from approximately 20 mM to 90 mM KCl (Figure 14), thus lowering the KCl concentration required to achieve optimal DNA polymerase activity as much as 6-fold. Further, SSB completely inhibited pol y when the concentration of KCl was greater than 150 mM. 79 Figure 13. W SSB stimulates the rate of DNA synthesis by W pol y. The rate of DNA synthesis was determined as described under "Methods" in the presence of the indicated amounts of E. coli SSB and 30 mM KCl (open circles) or 120 mM KCl (closed circles). 80 Figure 13 700 “ th cs .>9—>HHU< Mm40m 81 Figure 14. mu SSB lowers the KC! optimum of Drosophila pol y. The rate of DNA synthesis was determined as described under "Methods" in the presence of the indicated amounts Of KCl and in the absence (closed circles) or presence (Open circles) of lug of SSB. 82 150 200 100 KCl, mM id . . u . . 4 0 0 0 0 0 0 0 0 0 8 6 4 2 Chm as .>.:>=.U< Hm46m Figure 14 83 SSB stimulation of DNA polymerase 7 activity exhibits a lag time. E. coli SSB stimulates bacteriophage T7 DNA polymerase up to 11-fold following two minutes of DNA synthesis, decreasing with time down to 5-fold after 15 minutes of catalysis (Myers and Romano, 1988). To determine the maximal stimulation of pol 'y by SSB, we examined the extent of DNA synthesis by pol y in the absence and presence of SSB over a time course from 1 to 60 minutes in the presence of 30 mM KCl. Incorporation of nucleotides by pol 7 was linear up to about 60 minutes of incubation at 30'C (Figure 15A and Kaguni et al., 1988), while incorporation by pol y in the presence of SSB was linear only from 4 to 16 minutes of incubation. Stimulation of DNA polymerase was 7-fold initially (0.1% of DNA replicated), then over 30-fold from 4 to 16 minutes (2-9% of DNA replicated), but decreased to 17-fold follOwing 60 nrinutes (18% of DNA replicated) of incubation at 30'C (Figure 15B). In comparison, the 25-fold stimulation of polymerase activity observed in previous experiments was achieved following 30 minutes of incubation (13% of DNA replicated). Thus, a 4 minute lag is required to attain maximal stimulation of pol y by E. coli SSB of 30- to 35-fold in the presence of 30 mM KCl. SSB has little effect on the processivity of DNA polymerase ‘y. T he stimulation of Drosophila mitochondrial DNA polymerase by E. coli SSB may be due to its ability to increase the processivity, rate of initiation of DNA synthesis, or intrinsic rate of nucleotide incorporation by y polymerase. Alternatively, SSB may increase DNA polymerase cycling or stimulate pol 7 activity by some combination of all of these parameters. In an initial effort to determine the mechanism by which SSB stimulates pol 7 activity, its effect on enzyme processivity was examined at 30 mM KCl (Figure 16). The addition of SSB resulted in a more uniform distribution of DNA synthesis products, likely indicative of the elimination of some DNA polymerase pause sites, and a 2- to 3-fold increase in the amount of full length DNA synthesis products apparent. However, the processivity of mitochondrial DNA polymerase was increased less than 3-fold after 8 84 Figure 15. M SSB stimulation of Drosophila pol yexhibits a lag time. The rate of DNA synthesis was determined over a time course as described under "Methods" in the presence of 30 mM KCl and in the absence (closed circles) or presence (open circles) of 1 pg of SSB. A. Nucleotide incorporation by pol 7 was determined following 0, 1, 2, 4, 8, 16, 30, 45, and 60 minutes Of incubation. Following 60 minutes and in the absence of SSB, 1% of the DNA available was copied, while in the presence of SSB, 18% of the DNA was copied. B. The data from "A" was replotted as the extent of stimulation resultant from the presence of SSB at each time point. 85 60 50 30 40 TIME, MINUTES 20 10 . . q . . . . 4 0 0 0 0 0 8 6 4 2 100 " Gm9 $4025. ZC >._._>_._.U< mwAOm Z ENZYME PURITY. FRACTION 0 123 456 6557 — 4361 — 1925 — 1207 —~ 272 — 176 — 125 — Figure 21 120 of pol y, we examined the ability of the stimulatory fractions to bind to template-primer DNA (LSK-7, see figure 20) by a gel electrophoretic mobility shift (gel retardation) analysis. DNA binding was observed in all fractions assayed (18-52): Fractions 18 and 20 _ contained a DNA binding activity which shifted the template DNA to a position similar to that of purified pol 7 (Figure 22B, compare lane P with lanes 18 and 20). Fractions 22-52 contained a DNA binding activity responsible for shifting the template DNA to a more slowly migrating position than pol 7, which generally correlated with DNA polymerase stimulatory activity. Finally, fractions 3246 contained a third DNA binding activity, likely identical to a mtDNA binding activity previously identified (D.L. Lewis and LS. Kaguni, unpublished observation), which shifted the template DNA to a much more quickly migrating position than pol 7. SDS-PAGE analysis of the stimulatory fractions revealed that of approximately fifteen polypeptides present, only about six could be responsible for the DNA binding and DNA polymerase stimulation Observed. However, a 145 kDa polypeptide, representing greater than 90% of the total protein present, was evident in fractions 18-52, peaked in fractions 24-30 and appeared to be the most likely source of DNA binding and polymerase stimulatory activities (Figure 22C). DNA binding and DNA polymerase stimulatory activities are separated by gel filtration chromatography. In order to obtain further purification of protein exhibiting these activities and to determine its native molecular mass, peak fractions were chromatographed on an Sephacryl S-200 gel filtration column, resulting in the separation of the DNA binding and DNA polymerase stimulatory activities (Figure 23). While DNA binding to template-primer DNA was evident in fractions 28-36, DNA polymerase stimulatory activity eluted in fractions 34-46. Comparison of the elution positions of peak fractions with those of known protein standards allowed us to estimate the Stokes radii of the two activities. DNA polymerase stimulatory activity had a Stokes radius of 43 A, 121 FIGURE 22. Co-sedimentation of DNA polymerase stimulatory and DNA binding activities. A. DNA polymerase activity (open circles) was determined on singly-primed M13 DNA in the presence of 120 mM KCl, while DNA polymerase stimulatory activity (closed circles) was determined on singly-primed M13 DNA in the presence of 30 mM KCl. In the absence of stimulatory fractions, pol 7 activity was 6.0 Wm]. B. DNA binding activity on LSK-7 was determined as described under "Methods". Lane P indicates addition of pol y. Lanes 18-52 correspond to the glycerol gradient fractions assayed. C. Stimulatory and DNA binding fractions were analyzed by SDS-PAGE and silver staining. Lanes 18-52 correspond to the glycerol gradient fiactions assayed. Fraction 122 0 10 20 30 40 50 60 POLYMERASE ACTIVITY, U/ML “th113“ .- - i 2‘ U balm?!” Figure 22 123 FIGURE 23. Gel filtration of DNA polymerase stimulatory and DNA binding activities. DNA binding activity on LSK-7 (open circles) was determined and quantitated as described under "Methods". Bindng activity was plotted as a function of the optical density of each shifted complex (integrated optical density (IOD)). DNA polymerase stimulatory activity (closed circles) was determined on unprimed M13 DNA as described under "Methods". 124 ZO~H pol shift—> Figure 24 128 humans, the DNA binding activity may be specific for template-primer DNA. Gel retardation and filter binding studies indicate that replication factor-C binds template- primer, but not sin gle-stranded or double-stranded DNA (Lee et al., 1990; Tsurimoto and Stillman, 1990). Further, footprints of RF-C and T4 gp44/62 indicate that they bind to the template-primer junction (Munn and Alberts, 1991a; Tsurimoto and Stillman, 1991). In contrast, the E. coli processivity factor, pol III B subunit, does not bind DNA in the absence of its primer recognition complex, but once loaded onto the DNA, binding is stable and non-specific (Stukenberg et al., 1991). Production of polyclonal antiserum raised against the 145 kDa polypeptide. Further characterization of this putative template-primer specific DNA binding activity required 145 kDa polypeptide of higher purity and in greater abundance. Because the purity and yield of this polypeptide varied in several DNA polymerase y preparations (data not shown), a purification scheme developed specifically for the DNA binding activity from mitochondrial extracts was desired. However, because we were unable to assay crude mitochondrial fractions for DNA binding activity due to nuclease degradation of the template DNA, we chose to produce polyclonal antiserum against the 145 kDa polypeptide for use in its purification. Due to the impurity of the available DNA binding fractions, the 145 kDa region of an SDS gel was excised and the protein eluted or the gel slice ground finely in preparation for injection. The resultant serum, but not the preimmune serum, recognized the injected material and 2-3 polypeptides of approximately 145 kDa in pol 7 fraction HI (z7% pure) in an immunoblot analysis (Figure 25). Purification of the 145 kDa putative DNA binding protein. In order to confirm the relationship between the DNA binding activity and the 145 kDa polypeptide, estimate the pol 'y to DNA binding activity stoichiometry, and to further characterize this activity, we undertook the purification of the 145 kDa polypeptide using an immunoblot assay. Initial 129 FIGURE 25. Reactivity and specificity of polyclonal antiserum raised against the 145 kDa polypeptide. P01 7 fraction III (1.4 ug total protein; lanes 1 and 2) and VI (; 100 ng total protein; lane 3), and glycerol gradient purified 145 kDa polypeptide (200 ng total protein; lane 4) were electrOphoresed on an 8% SDS gel, transferred to nitrocellulose and probed with antiserum raised against the 145 kDa polypeptide (lanes 1 and 4), preimmune (lane 2), or antiserum against pol 7 (lane 3). Antiserum against pol y detects the pol yor- subunit (125 kDa), while antiserum against the 145 kDa polypeptide detects slightly larger polypeptides estimated to be about 145 kDa. 130 145 kDa protern — 4f _. 145 kDa protein '1 125 kDa j polymerase subunit Figure 25 13 1 efforts were directed at following the 145 kDa polypeptide through the mitochondrial DNA polymerase purification to determine the level of co-purification of the two proteins. Examination of chromatographic fractions eluted from phosphocellulose, the initial column of the pol y purification, revealed that less than 10% of the 145 kDa protein was present in the pol 7 peak fractions, and that it is approximately 20-fold more abundant than 7 polymerase at this stage of the purification. Immunoblots of subsequent pol 'ypurification steps showed that the 145 kDa protein co-purified almost quantitatively with the DNA polymerase over three columns before resolving from y polymerase during glycerol gradient sedimentation (data not shown). Purification of the majority of the 145 kDa protein, which separated from pol 7 during chromatography on phosphocellulose, was achieved by following the procedure for mitochondrial DNA polymerase purification (Wernette and Kaguni, 1986): from 200 g of Drosophila embryos, approximately 150 11g Of the 145 kDa protein (>90% pure) was Obtained. In comparison, this purification yields 10 ug of pol y (Wernette and Kaguni, 1986) and a similar purification yields 500 11g of pol or (Kaguni et al., 1983a). Although DNA binding activity was detectable, nuclease modification of the template DNA was evident even at this stage of the purification (data not shown). Therefore, approximately 25 pg of the 145 kDa protein was chromatographed on a Sephacryl S-200 gel filtration column to yield several polypeptides of approximately 145 kDa, comprising greater than 95% of the protein detectable by SDS-PAGE and silver staining (Figure 26A). Although multiple polypeptides are present, they appear to be due to proteolysis as they are all detected by the antiserum raised against the 145 kDa protein and became more prevalent as the purification proceeded (data not shown). Further, DNA binding analysis revealed that a DNA binding activity which chromatographs closely with the 145 kDa protein is present in these fractions (Figures 26B and C). Thus, the 145 kDa protein is responsible for the observed template- primer specific DNA binding, and has been designated DNA binding protein-145 (DBP- 145). 132 FIGURE 26. Co-chromatography of DNA binding activity and 145 kDa polypeptide. A. DNA binding fractions were electrophoresed and the protein stained with silver. Lanes 15-41 correspond to the gel filtered fractions assayed. B. DNA binding to LSK-7 was determined as described under "Methods". No protein was added to lane 0. Lane L is the load and lanes 15-41 correspond to the gel filtered fractions assayed. C. Quantitation of SDS-PAGE and DNA binding analyses was determined as described under "Methods". Protein levels (closed circles) and bindng activity (open circles) were plotted as functions of the optical density of each stained or shifted complex (integrated optical density (IOD)). 133 1517 19 21 23 2527 2931 33 35 37 3941 205 — 116— 97— 45— 0 L151719 2123 25 27 29 313335 37 39 41 he. i iiii i‘hiiiiii'i C 50 ' 5 :1 9.. <5 E G .7: at: < Z Q 10 20 so 40 FRACTION 145 kDa PROTEIN, IOD 134 Physiological role of DNA binding protein-145. Preliminary experiments utilizing glycerol gradient purified DBP-145 indicated that there was no detectable DNA or RNA polymerase, DNA primase, topoisomerase, helicase, ligase, or ATPase activities associated with it (data not Shown). In addition, DBP-145 antiserum does not recognize Drosophila DNA polymerase or, y, 8, topoisomerase I or II, human RF-C, or yeast mtRNA polymerase in immunoblot analyses (C.L. Farr, data not shown). Further, antiserum raised against Drosophila DNA polymerase (I, y, topoisomerase 11, human RF-C, or yeast mt RNA polymerase does not recognize DBP-145 in an immunoblot (C.L. Farr, data not shown). . Elucidation of the physiological role of DBP-145 could likely be attained through a careful survey for all activities known to bind DNA during replication and transcription. However, we chose to obtain arrrino acid sequence of the protein for several reasons. First, because of our uncertainty of its mitochondrial origin, and because several enzymes involved in cellular DNA metabolism have been identified in Drosophila, it would allow us to determine immediately if DBP-145 is one of these proteins. In addition, since we have purified DBP-l45 by immunoblot, it is possible that even though DNA binding activity is apparent, it has not retained any other associated activities. Therefore, conventional assays for these putative activities may nor be useful. Finally, the generation of PCR primers from protein sequence should allow us to obtain a cDNA clone of DBP- 145 and use the derived nucleotide sequence to identify conserved sequence elements which may lead to a suggested role for this protein. Initial efforts at obtaining internal amino acid sequence have been hampered by the concurrent sequencing of multiple peptides generated by proteolytic cleavage with endo Lys c. However, sequence of four proteolytic fragments, totalling 51 anrino acids, has revealed that DBP-145 does not exhibit identity to any sequence available for search by the TFastA program of the GCG Package, Version 7 (D.L. Lewis, personal communication). 135 DISCUSSION Our attempts to determine reaction conditions in which mitochondrial DNA polymerase could copy DNA with high efficiency and processivity were unsuccessful: reagents which increased the processivity of the enzyme invariably decreased its activity as well. In contrast, E. coli SSB increased the rate while maintaining the high processivity of DNA synthesis by pol y. In an effort to identify mitochondrial protein factors which might enhance the efficiency of DNA synthesis by y polymerase, we examined the ability of partially purified fractions of pol 'y to copy singly-primed single-stranded DNA. Although its processivity remained unchanged, the rate of DNA synthesis by mitochondrial DNA polymerase was diminished upon glycerol gradient sedimentation, Suggesting the loss of a DNA polymerase accessory factor. A putative accessory factor for DNA polymerase a, C1C2, had similar characteristics: it had no effect on the processivity of pol a, but stimulated activity depending on the primer density of the template DNA (Pritchard et al., 1983). In contrast, the primer recognition factors associated with E. coli pol III and bacteriophage T4 DNA polymerase increase DNA polymerase activity and processivity ' (Newport et al., 1980; Huang et al., 1981, Pay et al., 1982; LaDuca et al., 1983), while that associated with human pol 8 had no effect on either enzymatic characteristic (Lee and Hurwitz, 1990; Lee et al., 1991). Further analysis of the glycerol gradient fractions revealed that DNA polymerase stimulatory activity appeared to co-sediment with a DNA binding activity and a 145 kDa polypeptide. Purification of the glycerol gradient fractions by gel filtration resulted in the resolution of the DNA binding and DNA polymerase stimulatory activities. The 145 kDa polypeptide eluted with DNA binding activity, while nuclease activity eluted in the position of DNA polymerase stimulatory activity. The stimulatory effects of nuclease on in vitro DNA polymerase activity are well documented: their ability to produce additional primer termini by partially degrading dsDNA or facilitating overlapping of ssDNA renders inert 136 templates active for DNA polymerase activity (Richardson et al., 1963; Lehman, 1967; Kornberg and Baker, 1992). Thus, our original identification of a factor which stimulated mitochondrial DNA polymerase activity appears to have been misleading. However, further characterization of the DNA binding activity was attempted. Combining the data from gel filtration and sedimentation analyses allowed us to calculate a native molecular weight of 112 kDa for the DNA binding activity, well below the suspected molecular weight of 145 kDa obtained from SDS-PAGE. However, this calculated molecular weight is likely an underestimate due to the poor resolving power in this region of elution from the gel filtration column, as evidenced by the disparate sizes of proteins which eluted with the DNA binding activity (bovine liver catalase (230 kDa) and glutamate dehydrogenase (340 kDa)). Although it may seem surprising that a 145 kDa polypeptide would filter with these larger proteins, it is consistent with the sedimentation results and is indicative of a protein which displays a high degree of asymmetry. Proteins sediment in proportion to their molecular weight and inversely proportional to their frictional coefficient, a function of molecular shape: the further a molecule deviates from a sphere, the higher its frictional coefficient (Cantor and Schimmel, 1980; Zubay, 1988). Therefore, asymmetric molecules sediment slower than spherical molecules and appear to be smaller than they actually are ' (the DNA binding activity sediments with a spherical marker of 68.5 kDa). In contrast, asymmetric molecules filter faster than spherical molecules and appear larger than they actually are (the DNA binding activity activity filters with spherical markers of 230 kDa and 340 kDa). Calculations of molecular weight obtained by combining these two methods negates these deviations due to asymmetry (Siegel and Monty, 1966). Therefore, because of the suspected underestimate of Stokes radius by gel filtration, the DNA binding activity appears to be an asymmetric protein larger than 112 kDa. Consistent with this hypothesis the frictional coefficient for the DNA binding activity was calculated to be 1.7, whereas "spherical" proteins exhibit frictional coefficients up to only 1.4 (with a sphere having a coefficient of 1.0; Creighton, 1984). 137 Like primer recognition factors from E. coli, bacteriophage T4 and humans, DBP- 145 appears to be a template-primer specific DNA binding protein. DNA footprinting studies Show that bacteriophage T4 gp44/62 and human RF-C bind the template-primer junction, covering about 20 base pairs and extending 3-4 nucleotides past the junction onto the template Strand (Munn and Alberts, 1991a; Tsurimoto and Stillman, 1991). In addition, these factors exhibit a preference for primed template DNA as the cofactor for DNA dependent ATPase activity (Jarvis et al., 1989b; Tsurimoto and Stillman, 1990). Similarly, E. coli 7 complex appears to bind the template-primer junction and requires primed template as a cofactor for its ATPase activity (Griep and McHenry, 1991; Onrust et al., 1991). The lack of binding by DBP-145 and pol y to ssDNA appears contradictory to their purification on ssDNA cellulose and to our proposal that SSB eliminates non- productive binding of pol y to single stranded template DNA. However, these DNAS likely form extensive secondary structure, resulting in regions of dsDNA and apparent template- primer junctions, that serve as binding sites. In fact, putative secondary structure in unprimed M13 DNAS of natural sequence was proposed to be an effector of the E. coli 7 complex ATPase (Onrust et al., 1991). Thus, our results are consistent with the possibility that DBP-145 is a primer recognition factor similar to those from E. coli, bacteriophage T4, and humans. However, we cannot exclude the possibility that DBP-l45 is a protein involved in DNA replication or transcription in some role other than as a DNA polymerase accessory factor even though we detected no enzymatic activity associated with DNA binding. Antiserum raised against the 145 kDa polypeptide recognizes DBP-145 following its resolution from pol 7 upon glycerol gradient sedimentation and partially purified DBP- 145 present in pol 7 Fr III (~7% pure), but does not recognize Drosophila pols a, 'y, 8, topoisomerase I, II, human RF-C, or yeast mitochondrial RNA polymerase in immunoblots (C.L. Farr, data not shown). Nor do antibodies raised against Drosophila pol or and topoisomerase II, human RF-C, or yeast mitochondrial RNA polymerase 138 recognize DBP-l45. Thus, DBP-145 is neither pol or, y, or 8 nor topoisomerase I or II, but may be Drosophila homologs of RF-C or mtRN A polymerase. Further, because the immune serum is specific for DBP-145 in crude fractions, it was an excellent candidate for use in immunoblots to assay for DBP-145 purification even though the resultant protein would potentially be partially or wholly inactive. Purification of DBP-145 yielded a 145 kDa polypeptide which exhibited template-i primer DNA binding activity, and was greater than 95% pure and approximately 15- to 20- fold more abundant than pol y. DBP-145 may be involved in mtDNA transcription as it appears to be approximately equal in abundance to mtRNA polymerase in protein immunoblots of crude fractions. Alternatively, DBP-145 may be a protein involved in mtDNA replication that is present in mitochondria at greater levels than pol y is: proteins involved in DNA replication in E. coli and bacteriophage T4, including DNA helicase (Venkatesan et al., 1982; Richardson and Nossal, 1989), topoisomerase (Kaguni and Kornberg, 1984; Funnel et al., 1987; Baker and Kornberg, 1988; Baker et al., 1986; Baker et al., 1987), and ligase (Kornberg and Baker, 1992) are up. to 500-fold more abundant than replicative DNA polymerase. Although inconsistent with roles as mitochondrial DNA helicase (Lahaye et al., 1991), topoisomerase I (Brun et al., 1981; Lazarus et al., 1987), origin recognition and regulatory proteins (Barat et al., 1985; Mignotte and Barat, 1986; Qureshi and Jacobs, 1993), or transcription factors (Fisher and Clayton, 1985; Diffley and Stillman, 1991), the size of DBP-145 suggests that it may be mitochondrial topoisomerase II (Melendy and Ray, 1989), primase (Wong and Clayton, 1985a; Ledwith et al., 1986), ligase, or RNA polymerase (Kelly and Lehman, 1986; Bogenhagen and Insdorf, 1988) even though we were unable to detect any Of these enzymatic activities. However, antiserum against the yeast mtRNA polymerase does not recognize DBP-145 in an immunoblot, but does detect a 2:150 kDa polypeptide in Drosophila mitochondrial fractions which co-chromatographs with RNA synthetic activity (C.L. Farr and LS. Kaguni, 139 unpublished observations). Thus, it is unlikely that DBP-145 is Drosophila mtRNA polymerase. Amino acid sequence obtained from four proteolytic peptides derived from DBP- 145 revealed that it exhibited no identity with any sequence in libraries searched by the GCG TFastA program. This lack of identity does not exclude the possibility that DBP-145 is a component of the Drosophila mitochondrial DNA replicational or transcriptional enzymatic machinery Since these proteins have not been sequenced. The use of these peptide sequences to produce degenerate oligonucleotides as primers for PCR amplification and sequencing of cDNA encoding DBP-145 should allow us to hypothesize a role of DBP-l45 in DNA metabolism. We have purified a 145 kDa protein which displays template-primer DNA binding specificity but no Other detectable enzymatic activities. Although it was originally identified as a putative DNA polymerase accessory factor, it has no effect on the activity or processivity of mitochondrial DNA polymerase. The enzymatic characteristics of pol y in the presence of 30 mM KCl and E. coli SSB are consistent with those predicted by models of in vivo mitochondrial DNA replication. Although mitochondrial SSB has been identified and is required in vivo for yeast mitochondrial DNA replication, additional DNA polymerase accessory factors need not be necessary. DNA polymerase 7 has been identified as a member of the family A DNA polymerases which comprises enzymes that exhibit homology to E. coli DNA polymerase 1, including bacteriophage T7 DNA polymerase '(Ito and Braithwaite, 1991). Bacteriophage T7 DNA polymerase is a heterodimer comprising an 80 kDa catalytic core, exhibiting 5'-)3' DNA polymerase and 3'-—>5' exonuclease activities, and an 11 kDa accessory factor (Adler and Modrich, 1979; Hori et al., 1979a, b). Separation of the two subunits results in diminution of polymerase activity to only 0.5-2% of normal activity (Adler and Modrich, 1979). Sirrrilarly, attempts to separate the subunits of pol 7 led to loss of enzyme activity (M.W. Olson and LS. Kaguni, unpublished observations). The accessory factor of T7 DNA polymerase is the 140 host (E. coli) thioredoxin protein and serves to increase DNA polymerase processivity approximately 50-fold to greater than 5000 nucleotides (Huber et al., 1986; Tabor et al., 1987). T7 DNA helicase-primase has been proposed to function as the primer recognition activity for T7 DNA polymerase (Nakai and Richardson, 1986 a, b), although it is not required for processive DNA synthesis. Additional similarities between mitochondrial and T7 DNA replication are evident: the origin of replication of T7 DNA has two RNA polymerase promoters, like the mitochondrial origin, and a 61 base pair A+T region (Saito et al., 1980; Tamanoi et al., 1980; Clayton, 1991). Origin recognition and template melting of both is proposed to occur thrOugh transcription by their respective RNA polymerases, and the 3' ends of these transcripts prime leading strand DNA replication (Itoh and Tomizawa, 1980; Chang and Clayton, 1985; Chang et al., 1985; Fisher et al., 1987). Further, yeast mtRNA polymerase appears to be related to T7 RNA polymerase (Masters et al., 1987 ). Thus the heterodimeric mitochondrial DNA polymerase may, like T7 DNA polymerase, comprise a catalytic core and a processivity factor which does not require an ATP dependent loading mechanism. The cloning and overexpression of mtDNA polymerase should help to address this issue. CHAPTER V SCREENING A cDNA EXPRESSION LIBRARY FOR MITOCHONDRIAL DNA POLYMERASE FROM DROSOPHILA 141 142 INTRODUCTION Putative accessory proteins to mitochondrial DNA polymerase have proven to be difficult to identify and purify, in part due to the lack of a highly Specific functional assay. The generation of such an assay may be dependent on the development of an in vitro replication system requiring all of the enzymatic and nucleic acid participants in mitochondrial DNA replication. Our efforts to develop simpler assays to examine activities which contribute to the efficiency of mtDNA replication (e. g. Stimulation of DNA polymerase activity, processivity, and primer binding) have been hindered by the modification of DNA substrates due to the presence of nucleases in even partially purified fractions. The use of DNA polymerase affinity purification to identify 7 polymerase accessory factors would alleviate many of the problems introduced by the presence of nucleases in crude fractions. Here, mitochondrial DNA polymerase would be coupled to a chromatographic matrix, to which crude a mitochondrial extract would be applied: polypeptides which exhibited affinity for DNA polymerase would be candidates for polymerase accessory proteins. The identification and purification of DNA polymerase accessory proteins by this method has proven successful in the case of bacteriophage T4 accessory factors (Formosa et al., 1983), but requires a level of DNA polymerase approximately 15,000-fold higher than that obtained from a typical preparation Of D. melanogaster mitochondrial DNA polymerase performed in our laboratory (W emette and Kaguni, 1986). Cloning and overproduction of the cDNAs encoding the subunits of DNA polymerase 'y will provide the levels of protein necessary to use this strategy. Given the possible benefits of the cloning and overproduction of DNA polymerase y we screened a Agtll cDNA expression library, using antiserum against pol y as a probe (Young and Davis, 1983). We chose this method due to the immediate availability of polyclonal antiserum and the impetus that this procedure had been used successfully with 143 the same cDNA library for a protein of similar size, DNA topoisomerase II (Nolan et al., 1986). 144 EXPERIMENTAL PROCEDURES MATERIALS lgtll cDNA library. The Agtll cDNA library was obtained from the laboratory of Tao-shih Hsieh of Duke University and its preparation has been described (Nolan et al., 1986). Briefly, mRNA was isolated from Drosophila melanogaster embryos and cDNA was prepared. EcoRl linkers were added and the cDNAs were inserted into the single EcoRI site of the hgtll expression vector. The average insert size was 800 base pairs. Nucleic Acids. Template DNA for Polymerase Chain Reactions (PCR) was prepared from A phage plate lysates. 1.. phage were treated with DNase I and RNase A (10 [lg/ml each), PEG precipitated, phenol and chloroform extracted, treated with SDS (2%) and proteinase K (200 rig/ml), re-extracted with phenol and chloroform, and ethanol precipitated. Primers complementary to the Agtll DNA sequence, adjacent to the EcoRl cloning site, for use in PCR were obtained from Dr. Zachary Burton of this department: sense primer 5'-GGTGGCGACGACTCCTGGAGCCCGTCAGTATCGGCG-3', anti- sense primer 5'-TTGACACCAGACCAACTGGTAATGGTAGCGACCGGC—3'. Enzymes. Sequenase version 2.0 was from United States Biochemical. METHODS Screening the ’1ng cDNA library. Screening was carried out as described (Young and Davis, 1983). The Agtll library was screened with antibody probes as plaques on a lawn of E. coli Y1090 ((ATCC no. 37197) = E. coli AlacU 169 proA+ Alon araDlB9 strA supF[trpC22::Tn10] (pMC9) pMC9 = pBR322-lacIQ). Infected E. coli was plated and grown under lytic conditions until plaques formed, IPTG impregnated nitrocellulose filters were overlaid and plates were further incubated to allow adhesion of the fusion protein. Filters were removed, washed, and probed with polyclonal serum against pol y and subsequently detected with protein A-alkaline phosphatase. Prior to use in the screening, pol 7 antibodies were preadsorbed with filter immobilized total proteins from an E. coli 145 Y10901ysate, resulting in removal of most anticoliform antibodies and giving significantly lower background in subsequent screening of plaques. Antibody affinity purification. Plaque purified Agtll stocks were used to prepare plate lysates. Screening was carried out as above except filters were probed with polyclonal antiserum against pol y, washed, and incubated for 10 minutes in 100 mM glycine, pH 3 to elute the bound antibodies. BSA and his base were added to 1% and 50 mM final concentrations, respectively, to neutralize the solution. These antibody solutions were subsequently used in western blots to detect pol y. Polymerase chain reaction analysis of insert size. PCR reactions (0.1 ml) contained 50 mM Tris-HCl, 1.5 mM MgC12, 20 mM (NI-I4)2SO4, 160 M dNTPs, 100 ng template DNA, and 1 11g each of primers complimentary to lgtll DNA adjacent to the EcoRI ' cloning site. Samples were overlaid with 75 in light mineral oil and heated at 95°C for 5 mins. Replinase (2 units; DuPont-NEN) was added while the reactions were still hot, and the PCR cycle profile was initiated: 94'C for 1 min. and 72°C for 10 mins. After 25 cycles, the reactions were incubated an additional 7 mins. at 72°C and stored at 4°C. Aliquots of each reaction were made 80% in formamide, 90 mM in Tris-borate (pH 8.3), 0.1% in xylene cyanol, and 0.1% in bromphenol blue, denatured for 2 mins. at 100°C and separated on a 6% polyacrylamide slab gel (13 x 16 x 0.15 cm) containing 7 M urea in 90 mM Tris-borate (pH 8.3) and 25 mM EDTA. DNA was visualized by staining in ethidium bromide (1.5 llg/ml) for 30 min. and washing in water for 20 mins. Sizes of PCR products were determined by comparison to I-IpaII restriction fragments of MlBGoril replicative form DNA (Kaguni and Ray, 1979) and HindIII restriction fragments of A DNA that were electrophoresed in adjacent lanes. Overproduction and sizing of B—galactosidase-pol yfusion protein. A crude lysate was prepared as described (Huynh et al., 1984) from a kgtll recombinant lysogen of Adm-771 constructed by Dr. Laurie Kaguni. The proteins in bacterial cell pellets were 146 separated by SDS-PAGE. Sizing of overexpressed fusion protein was by comparison to molecular weight markers (Sigma) run in an adjacent lane. Subcloning, DNA Sequencing and alignment with yeast 7 polymerase. PCR products of the 771 insert from Adm-771 were subcloned into M13Gori1 (Kaguni and Ray, 1979) by Dr. Laurie Kaguni and sequenced by Carol Farr using the dideoxy chain termination method of Sanger et a1. (1977) and Sequenase version 2.0. Sequence analysis was performed by David Lewis using the Program Manual for the GCG Package, Version 7, April 1991, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 5 371 1. 147 RESULTS Screening a Agtll cDNA library. The low abundance Of DNA polymerase 7 suggested that its corresponding mRNA levels may be low as well; therefore, the cDNA cloning was initiated by screening 4 x 107 plaque forming units (pfu) of a cDNA expression library constructed in lambda phage with polyclonal antiserum raised against pol 7. Primary screening yielded 140 putative positives, 43 of which were carried through subsequent screenings, finally yielding l8 putative pol yclones. Verification of putative positive clones. The use of a probe derived from crude polyclonal antiserum introduced the possibility that the phage identified as containing pol 7 clones may express polypeptides recognized by antibodies present in the serum other than those specific for y polymerase. Therefore, verification of the plaque purified clones was performed by affinity purifying antibodies reactive with the putative B-gal : pol 7 fusion proteins and subsequently testing the reactivity of these antibodies to pol 7 (Fr III, 7% pure) in a protein immunoblot. Antibodies purified from only one (Adm-W 1) of the final 18 putative positives reacted strongly with the (ll—subunit of mitochondrial DNA polymerase (Figure 27). Analysis of Adm-171. Overproduction of Adm-W1 fusion protein (data not shown) allowed us to assign this putative cDNA clone a size of =300 base pairs (bp), in good agreement with PCR analysis of the insert Size of 380 bp. The insert was subcloned into a bacteriophage M13 vector by Dr. Laurie Kaguni and sequenced by Carol Farr. Alignment of the 337 bp insert of Adm-Y7 l by David Lewis to the catalytic subunit of yeast mitochondrial DNA polymerase (Foury, 1989) revealed that it exhibited 24% identity and 45% similarity to yeast pol 7 (Figure 28A): the putative pol 7 clone aligns with sequence outside of the conserved regions common to DNA polymerases (Figure 28B). Shuffling of 148 Figure 27. Reactivity of fusion protein afiinity-purified antibodies with Waste: DNA polymerase 7. Antibodies were affinity purified as described in the methods. P01 7 Fraction III (6.4 units, 1.4 ug total protein; lanes 1 and 2) and Fraction VI (8 units, 100 ng total protein; lanes 3 and 4) were denatured, electrOphoresed in a 10% SDS-polyacrylamide gel and transferred to nitrocellulose. The blot was incubated with unfractionated rabbit antiserum against pol 7 (1:1000, lanes 1 and 3) or undiluted affinity purified affinity purified antibodies (lanes 2 and 4) for 3 hours at 25°C and then probed with protein A- alkaline phosphatase. 149 Figlne 27 150 Figure 28. Amino acid sequence alignment of Adm-771 with yeast mitochondrial DNA polymerase catalytic subunit. A. Anrino acid sequence was determined in forward and reverse in 3 reading frames and the best alignment shown here. Bars indicate identical matches and dots indicate conservative substitutions. Sequence from Adm-771 is numbered 1 through 84 and aligns with yeast pol ypositions 966 through 1078. B. The positions of exonuclease (exo) and polymerase (pol) conserved regions are indicated relative to the region of Adm-771 alignment. l 951 29 1001 58 1051 151 .........I ..... GEVID ...... .roexrvxsrrotorcouncptrr 28 II :I ...II I. :I:I : : NMDCITPSNKTAIPHGEALDINQLLDKPNSKLGKPSLDIDSKVSQYAYNY 1000 .IMDEIQKPIAR ....... ADELKNNKSDTKNI... ........ HFGA 57 :1 I REPVFEEYNKSYTPEFLKYFLAMQVESDKRDVNRLEDEYLRECTSKEYAR 1050 SAQNASLNIIBFNDDAQQDNSNQKKKN. ...................... 84 I ............ DGNTASYSLLDYIKDVEKGKRTKVRIMGSNFLDGTKNAKADQRIRLPVNM 1100 exo l l I DO I u_-___ l O I I — -1IHHFI ! I I 1 I 200 400 000 IN 1000 IfignutLZS 152 the sequence to determine the homology of random alignments revealed similar levels of identity and similarity to the preferred alignment (David Lewis, data not shown). 153 DISCUSSION Screening of a hgtll Drosophila cDNA expression library with antiserum against pol 7 yielded 18 putative DNA polymerase clones from 4 x 107 pfu screened. One of these 18 putative clones was confirmed as a D. melanogaster 7 polymerase clone due to its affinity for antibodies which were specific for the catalytic subunit of mitochondrial DNA polymerase. Further confirmation of this putative pol 7 clone was not obtained from its sequencing, as alignment with the yeast 7 polymerase catalytic subunit was outside conserved polymerase and exonuclease motifs. In order to estimate the expected frequency of obtaining a pol 7clone, we compared our results with those obtained from a similar screening of this same library for DNA topoisomerase II (topo H) cDNA. Here, putative topo II clones were obtained at a frequency of 1 in 106 pfu screened (Nolan et al., 1986), 10-fold more frequent than for pol 7. Given its role in nuclear chromosomal DNA replication, we can estimate that topo II is at least 50-fold more abundant than 7polymerase (Wernette et al., 1986). Thus, assuming that protein and mRNA levels between topo II and pol 7 are approximately proportional, it appears that we have Obtained a putative pol 7 clone at an appropriate frequency. Verification of the D. melanogaster putative pol 7 clones was performed by purifying reactive antibodies and examining their specificity for 7 polymerase in an immunoblot. Seventeen of the 18 putative clones exhibited affinity for antibodies which appeared to non-specifically (weakly) interact with either the (ll-subunit or both the OL- and B-subunits of pol 7 (data not shown). However, one putative clone, Adm-771, exhibited an affinity for antibodies which reacted specifically (strongly) with the catalytic subunit of pol 7. While it is possible that Adm-771 expresses a polypeptide which maintains an epitope in common with the a-subunit of pol 7, but is not itself pol 7, it is unlikely that antiserum against pol 7 would detect it since it does not recognize either E. coli DNA polymerase I or D. melanogaster DNA polymerase or (AK. Kolhoff, M.W. Olson, and 154 LS. Kaguni, unpublished observations) which might be expected to have antigenic determinants in common with pol 7. Mitochondrial DNA polymerase isolated from D. melanogaster is a 160 kDa heterodimer comprising 125 kDa (expected cDNA length of 3.3-3.4 Kb) and 35 kDa (expected cDNA length of 0.9-1.0 Kb) subunits. Although it is unknown whether unique mRNAs encode each subunit, it seems likely because bacteriophage and cellular DNA polymerases generally possess separate genes for individual subunits (Wong et al., 1988; McHenry, 1988). Sizing of the confirmed mitochondrial polymerase a subunit clone, Adm-771 by PCR and sequencing indicates that it is approximately 0.3 Kb in length: one tenth of the expected size of an (rt-subunit clone. Sequencing and alignment of Adm-771 with yeast mitochondrial DNA polymerase catalytic subunit (analogous to D. melanogaster mitochondrial DNA polymerase Ot-subunit), revealed only 24% identity and 45% similarity. While these levels of identity and similarity do not appear to be significant, alignment is observed outside conserved polymerase and exonuclease regions. Therefore, alignment neither provides confirmatory evidence nor eliminates the possibility that Mm- 771 is a mitochondrial DNA polymerase clone. Efforts are currently underway in our laboratory to rescreen the Agtll Drosophila expression library with putative pol 7 clone Adm-771. In addition, purified 7 polymerase will be used to obtain internal anrino acid sequence which will also be used either to screen the library or as a secondary screen for those clones which are recognized by Adm-771 to finally obtain full length mitochondrial DNA polymerase cDNA clones. CHAPTER VI SUMMARY AND PERSPECTIVES 155 156 We have examined the effects of reaction conditions, single-stranded DNA binding proteins, and enzyme purity on the mechanism of DNA synthesis by mitochondrial DNA polymerase from Drosophila embryos. The alteration of reaction conditions resulted in changes in the rate and processivity of DNA synthesis catalyzed by 7 polymerase. Preliminary experiments indicate that the 3'-)5' exonuclease activity and fidelity of nucleotide selection by pol 7 are also affected by alterations in reaction conditions (M.W. Olson, C.L. Farr, and LS. Kaguni, unpublished observations). Further characterization of these effects may facilitate our understanding of the coordinate action of the DNA polymerase and exonuclease activities as well as the contribution of the exonuclease activity to the fidelity of pol 7. Although the alteration of reaction conditions facilitated fully processive DNA synthesis by 7 polymerase, optimal DNA polymerase activity and processivity were not achieved concurrently: mitochondrial DNA polymerase always exhibited its highest rate of DNA synthesis at moderate salt and its greatest processivity at low salt. These results suggested that protein factors which increase the efficiency of DNA synthesis by pol 7 may be involved in mitochondrial DNA replication. The addition of E. coli SSB to a low salt 7 polymerase reaction resulted in a dramatic increase in DNA polymerase activity while the high processivity exhibited by pol 7 under these conditions was maintained. This increase in activity appears to be due to the ability of SSB to diminish non-productive DNA binding by pol 7, allowing each DNA polymerase molecule to actively engage in DNA synthesis. However, once it completes the replication of a DNA molecule, 7 polymerase appears to be slow in cycling to an unused primer terminus, although direct measurement of DNA polymerase cycling is required to confirm this hypothesis. In this experiment, DNA synthesis proceeds in the presence of a32P-TTP on singly-primed single-stranded wild type M13 DNA (6,407 nt). Following 16 minutes of incubation (the time we have estimated for complete loading of the DNA polymerase molecules) an excess of sin gly-primed single-stranded recombinant M13 DNA (10,650 nt) is added so that any DNA polymerase molecules which cycle will do so onto 157 the larger DNA. Following further incubation, DNA synthesis products are analyzed by native agarose gel electrophoresis. The amount of 10,650 nt product apparent is indicative of the amount of cycling that has occurred. We observed a four minute lag time prior to maximal stimulation of pol 7 by SSB which seems to be due to an inability of pol 7 to locate primer termini efficiently. Altering the conditions of preincubation may aid in determining the cause of this lag time. Possible variations include one where pol 7 is incubated in the presence of SSB-coated DNA and dNTPs and DNA synthesis is initiated by the addition of MgClz. Alternatively, 7 polymerase may require the incorporation of dNMPs to remain stably bound to the primer terminus. This hypothesis can be tested by comparing the lag time prior to maximal DNA polymerase stimulation using the above preincubation with one in the presence of SSB- coated DNA, MgClz and the dNTP required for initial incorporation. Here, DNA elongation would be initiated by the addition of the remaining three dNTPs. Further confirmation of our hypothesis of inefficient primer location by pol 7 may be acquired by altering the primer density of the template-primer DNA: an increase in primer density should decrease the lag time required to achieve maximal pol 7 stimulation by SSB. E. coli DNA polymerase III holoenzyme assembly occurs in two stages: in the rate limiting step, the DNA polymerase accessory factors form an ATP-activated preinitiation complex on a template-primer junction. Subsequently, pol III core locates and tightly binds this preinitiation complex (O'Donnell, 1987). Interestingly, following fully processive DNA synthesis of template DNA, pol III holoenzyme requires 1-2 minutes to cycle to an unused primer terminus (Burgers and Kornberg, 1982b). In contrast, the cycling time can be reduced to as little as 10 seconds if the acceptor template is already endowed with a preinitiation complex (O'Donnell, 1987). These data suggest that DNA polymerase accessory factors may be required for efficient DNA polymerase cycling in mtDNA replication. 158 Although we were unable to identify Drosophila mitochondrial SSB using its putative stimulation of DNA polymerase 7 activity, its identification and purification, as well as the characterization of its effect on pol 7 activity, are necessary in order to determine the physiological relevance of pol 7 stimulation by E. coli SSB. In addition, comparative studies using a heterologous mitochondrial SSB may support the conclusions drawn from the results using E. coli SSB. The abundance of nuclease activity in crude Drosophila mitochondrial fractions precludes the use of assays which require accurate detection of labelled DNAS. However, its characteristic size and chromatographic behavior should aid in the purification of mtSSB by less conventional means. Heat treatment of crude fractions may selectively inactivate nuclease activities over mtSSB activity. DNA binding proteins that are less than 20 kDa in size and bind very tightly to ssDNA cellulose may be good candidates for mtSSB. Further characterization of these putative mtSSBs may reveal a tetrameric structure and cooperative binding to ssDNA which are strong indicators of mtSSB-like proteins. Alternatively, Drosophila mtSSB may be recognized by antiserum raised against mtSSBs from other organisms. If so, then this cross-reactivity could be used to purify mtSSB by using immunoblots to assay crude fractions. Similarly, the use of southwestern DNA binding analyses may aid in identification of mitochondrial SSB as well as other mitochondrial DNA binding proteins. The high degree of sequence conservation between mtSSBs and E. coli SSB suggests that Drosophila mtSSB cDNA may be identified by using oligonucleotides generated from highly conserved regions of mtSSB amino acid sequence. In fact, the cDNA encoding human mtS SB was identified and cloned using sequence obtained from rat mtSSB (Tiranti et al., 1993’). Subsequent overproduction of mtSSB may allow characterization of its activities as well as facilitate our search for additional components of the mitochondrial DNA replicational and transcriptional machinery. The use of T4 single- stranded DNA binding protein (gp32) in protein affinity chromatography resulted in the preferential retention of at least eight proteins involved in DNA replication including the T4 159 DNA polymerase and primer recognition factor (Formosa et al., 1983). In addition, the eukaryotic single-stranded DNA binding protein, RF—A, interacts with DNA helicase in calf thymus cells (Zhang and Grosse, 1992), with SV40 T-antigen (DNA helicase; Melendy and Stillman, 1993), and with transcriptional activator proteins VP16, CAM, and p53 (Li and Botchan, 1993; He et al., 1993). During the course of purification, the ability of Drosophila mitochondrial DNA polymerase to copy singly-primed M13 DNA was enhanced then diminished, suggesting the loss of an accessory factor. While an apparent stimulatory factor resolved from p01 7 during glycerol gradient sedimentation, we found that it appeared to be associated with a potent nuclease activity. However, a template-primer specific DNA binding activity was also identified and purified to homogeneity. Unfortunately, we were unable to detect any enzymatic activities additional to DNA binding. Amino acid sequence analysis will allow us to construct oligonucleotides for use as primers for PCR, which we can use to obtain a portion of the cDNA encoding the DNA binding protein. Sequencing this DNA may result in the identification of conserved sequence elements, enabling us to develop a hypothesis for the enzymatic role of DBP-l45. Confirmation of this role will be obtained by carefully assaying for this activity and characterizing it in vitro. Screening a cDNA expression library with antiserum raised against Drosophila mitochondrial DNA polymerase resulted in the identification of a single clone verified by its ability to specifically retain antibodies which recognized the large subunit of pol 7 in a immunoblot. Amino acid sequence from pol 7 will be obtained and used in conjunction with this putative positive clone to rescreen the cDNA library in order to obtain a full length 7 polymerase clone. The overproduction of pol 7 will allow us to pursue identification and purification of mitochondrial DNA polymerase accessory factors through the use of DNA polymerase affinity chromatography. In addition, separate overexpression of the two subunits of 7 polymerase may allow us to assign a function to the small subunit. 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