Pharmac. Ther. Vol. 47, pp. 105-115, 1990 Printed in Great Britain. All rights reserved

0163-7258/90 $0.00 + 0.50 © 1990 Pergamon Press plc

Associate Editor: D. SHUGAR

THE ROLES OF D N A POLYMERASES ALPHA A N D DELTA IN D N A REPLICATION ROBERT W. TALANIAN* a n d GEORGEE. WRIGHT Department of Pharmacology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, U.S.A. Abstract--The identities and precise roles of the DNA polymerase(s) involved in mammalian cell DNA replication are uncertain. Circumstantial evidence suggests that DNA polymerase ct and at least one form of DNA polymerase 6, that which is stimulated by Proliferating Cell Nuclear Antigen, catalyze mammalian cell replicative DNA synthesis. Further, the in vitro properties of polymerases ct and 6 suggest a model for their coordinate action at the replication fork. The present paper summarizes the current status of DNA polymerases ct and 6 in DNA replication, and describes newly available approaches to the study of those enzymes.

CONTENTS 1. Introduction 1.1. Scope of this review 1.2. Mammalian cell DNA polymerases: a catalog 2. Roles of the Mammalian Cell DNA Polymerases in DNA Replication 2.1. DNA polymerases beta and gamma probably do not contribute to genomic DNA replication 2.2. A role for DNA polymerase alpha in DNA replication is firmly established 2.3. Role of DNA polymerase delta in DNA replication 3. Properties of DNA Polymerase Delta 3.1. DNA polymerase delta: a mammalian DNA polymerase with 3'--, 5' exonuclease activity 3.2. Enzymatic properties of DNA polymerase delta: comparison with DNA polymerase alpha 3.3. PCNA: a stimulatory cofactor of DNA polymerase delta 3.4. Structural relationship between DNA polymerases alpha and delta 3.5, Differential inhibitors of DNA polymerases alpha and delta 4. Why Might There be Two Replicative DNA Polymerases in Mammalian Cells? 4.1. Asymmetry at the replication fork 4.2. Hypothesis: DNA polymerases alpha and delta catalyze lagging and leading strand synthesis, respectively 4.3. Evidence for the asymmetric dimer hypothesis 4.4. Precedent for the asymmetric dimer hypothesis in prokaryotic systems 4.5. Precedent for the requirement of two polymerases in yeast DNA replication 5. Future Prospects 5.1. Role of DNA polymerase delta in mammalian cell DNA replication 5.2. Relationship between PCNA-sensitive and -insensitive DNA polymerase delta 5.3. Relationship between DNA polymerases alpha and delta Acknowledgements References

106 106 106 106 107 107 108 108 108 109 109 109 109 110 110 110 111 111 111 111

*Current address: Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, U.S.A. Abbreviations: Pol ~t, DNA polymerase ct; pol fl, DNA polymerase fl; pol 7, DNA polymerase 7; pol 6, DNA polymerase 3; PCNA, proliferating cell nuclear antigen; BuPdGTP, NZ-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate; SV40, simian virus 40; CHO, Chinese hamster ovary; BuAdATP, 2-(p-n-butylanilino)-2'-deoxyadenosine 5'-triphosphate; COMDP, carbonyldiphosphonate; pol III, E. coli DNA polymerase III; yeast pol I, yeast DNA polymerase I; yeast pol III, yeast DNA polymerase III. 105

106

R.V. TALANIANand G. E. WRIGHT 1. I N T R O D U C T I O N 1.1. SCOPE OF THIS REVIEW

An understanding of mammalian cell DNA replication will require an understanding of the properties and roles of the enzymes which catalyze the key reactions in that process. This review seeks to improve that understanding by summarizing the current status of the mammalian DNA polymerases thought to be involved in DNA replication. It does not discuss the roles in DNA replication of enzymes other than DNA polymerases, nor does it cover the enzymology of DNA repair or of mitochondrial DNA synthesis. 1.2. MAMMALIANCELL DNA POLYMERASES: A CATALOG There are at least four distinct enzymes in mammalian cells capable of catalyzing DNA polymerization, named in the order that they were discovered (Weissbach et al., 1975): DNA polymerase alpha (pol ct) (Bollum, 1960), beta (pol fl) (Baril et al., 197t; Weissbach et al., 1971; Chang and Bollum, 1971), gamma (pol 7) (Fridlender et al., 1972) and delta (pol 6) (Byrnes et al., 1976). The identity between mitochondrial DNA polymerase (Kalf and Ch'ih, 1968; Meyer and Simpson, 1968) and pol ], has been demonstrated (Bolden et al., 1977; Bertazzoni et al., 1977; Hiibscher et al., 1977). Two forms of pol 6 are known, differing in their sensitivity to stimulation by a protein called proliferating cell nuclear antigen (PCNA), referred to as PCNA-sensitive and -insensitive pol 6; pol 6 is the generic name for the two. 2. ROLES OF THE M A M M A L I A N CELL DNA POLYMERASES IN DNA REPLICATION 2.1. DNA POLYMERASESBETA AND GAMMA PROBABLY D o NOT CONTRIBUTE TO GENOMIC D N A REPLICATION

The roles of pol fl and pol 7 in mammalian cell DNA synthesis seem limited to the repair of genomic DNA and synthesis of mitochondrial DNA, respectively. The notion that neither enzyme participates in DNA replication derives mainly from a lack of the sort of positive evidence which implicates pol ~ and possibly pol d; in DNA replication. For example, Dresler and Kimbro (1987) found that 2',3'-dideoxythymidine 5'-triphosphate, a compound which inhibits pol fl and pol 7 with at least an order of magnitude greater potency than it does pol ~ or pol 6 (Edenberg et al., 1978; Waqar et al., 1978; Wahl et al., 1986), inhibited replicative DNA synthesis in permeabilized human fibroblasts with a potency reflecting that which it displays for pol ct and pol 6, rather than pol fl and pol 7. Conversely, aphidicolin (Brundret et al., 1972), a potent inhibitor of pol ct (Ikegami et al., 1978), and pol 6 (Lee et al., 1981; Goscin and Byrnes, 1982), which is without effect on pol fl and pol 7 (Ikegami et al., 1978), is capable of complete and potent inhibition of mammalian cell replicative DNA synthesis (Ikegami et al., 1978; Hanaoka et al., 1979; Berger et al., 1979; Huberman, 1981). The latter observations suggest that neither of the aphidicolin-resistant DNA polymerases, pol fl

and pol 7, participate in replicative DNA synthesis in vivo. A second line of evidence concerns the level of activity of the various DNA polymerases as a function of the stage of the cell cycle. One would expect that the activity of a replicative DNA polymerase would increase prior to S phase. The level of pol ~t activity positively correlates with the level of replicative DNA synthesis throughout the cell cycle (see below), but the levels of pol fl and pol 7 are independent of DNA replication (Chang et al., 1973; Fry and Loeb, 1986). Zmudzka et al. (1988) showed that the cellular level of pol fl mRNA also was independent of the cell cycle. Experiments using selective inhibitors of pol fl have supported the idea that the mission of that enzyme is DNA repair (Miller and Chinault, 1982a,b; Cleaver, 1983; Yamada et al., 1985), although experiments of the latter type are complicated by, for example, the participation of pol ct (Fry and Loeb, 1986) and pol 6 (Dresler and Frattini, 1986, 1988; Nishida et al., 1988; Dresler et al., 1988) in DNA repair, and the apparent ability of pol ~ and pol fl to cooperate or function redundantly in certain types of repair synthesis (Fry and Loeb, 1986). Pol t' is found in mitochondria (Bolden et al., 1977) and is most likely the only DNA polymerase in mitochondria (Bolden et al., 1977; Zimmerman et al., 1980). It is believed to be the only DNA polymerase involved in mitochondrial DNA synthesis (Fry and Loeb, 1986). Pol 7 also has been identified in nuclei (Bertazzoni et al., 1977; Hfibscher et al., 1977), although there is no positive evidence for a role for that enzyme in genomic DNA synthesis (Fry and Loeb, 1986). 2.2. A ROLE FOR D N A POLYMERASEALPHA IN D N A REPLICATION IS FIRMLY ESTABLISHED

The most firmly established role for any of the known mammalian DNA polymerases in DNA synthesis is that of pol c¢ in genomic DNA replication. Three major lines of evidence, summarized below, indicate that pol ct is a major or the sole replicative DNA polymerase in mammalian cells. (i) The level of pol :t activity in cells closely tracks the level of replicative DNA synthesis throughout the cell cycle (Fry and Loeb, 1986). Pol ~ activity is low in quiescent cells (Bertazzoni et al., 1976) and increases dramatically when cells are stimulated to divide (Chang and Bollum, 1972; Chang et al., 1973; Bertazzoni et al., 1976) or when synchronized cells enter S phase (Spadari and Weissbach, 1974; Chiu and Baril, 1975). Th6mmes et al. (1986) demonstrated by quantitative immunoprecipitation of pol ct at various stages of the cell cycle that the increase in the activity of the enzyme during S phase was accompanied by an increase in the synthesis of the enzyme. Wahl et al. (1988) showed that pol ct mRNA levels increased just prior to the peak of DNA synthesis in quiescent cells stimulated to divide, suggesting that regulation of pol ct gene expression is at the transcriptional level. (ii) Inhibitors ofpol ~ inhibit mammalian cell DNA replication (Fry and Loeb, 1986). Ikegami et al. (1978) presented evidence for the hypothesis that aphidicolin prevented cell division in sea urchin embryos as a result of inhibition of pol ~t rather

DNA polymerases alpha and delta than, for example, deoxyribonucleotide biosynthesis. Pedrali-Noy et al. (1980) later showed that aphidicolin was capable of synchronizing HeLa cells by blocking their entry into S phase. Unfortunately, in vivo studies with aphidicolin are ambiguous with respect to the roles of pol ct and pol 8 in DNA synthesis; after many such studies were performed, it was revealed that the compound inhibits the two enzymes with equal potencies (Lee e t a [ . , 1981; Goscin and Byrnes, 1982). The possibility that inhibition of DNA synthesis by aphidicolin is at least partially a result of inhibition of pol ~ in vivo is supported by the observations of Miller et al. (1985), who found that DNA synthesis in permeable human fibroblasts could be inhibited up to about 50% by the monoclonal antibody SJK 132-20 (Tanaka et al., 1982), a reagent which neutralizes pol ct but not pol & (Byrnes, 1985). It is also supported by the observations of Liu et al. (1983) and Liu and Loeb (1984), who isolated aphidicolin-resistant forms of pol ~ from aphidicolin-resistant mammalian cells. (iii) A mouse cell line which was temperaturesensitive in DNA replication yielded a temperaturesensitive mutant form of pol ct (Murakami et al., 1985; Eki et al., 1986, 1988).

107

replicative DNA synthesis in permeabilized Chinese hamster ovary (CHO) cells was BuPdGTP-resistant and aphidicolin-sensitive. Less direct studies also have supported the hypothesis that pol 8 participates in D N A replication. For example, Prelich et al. (1987a) have shown that the pol 8 cofactor PCNA is required for in vitro simian virus 40 (SV 40) replication; also, Wold et al. (1989) found that a PCNA dependent DNA polymerase, perhaps PCNA-dependent pol 8, was required for in vitro SV40 replication. Evidence for the involvement of PCNA in cellular DNA replication is provided by the observation of Liu et al. (1989) that inhibition of CHO cell PCNA biosynthesis, by means of introduction into cells of an antisense oligonucleotide complementary to PCNA mRNA, blocked entry of the cells into S phase. Finally, Marraccino et al. (1987) measured the activities of pol ct and pol 8 in crude extracts of CHO cells by the susceptibility of the DNA polymerase activity to inhibition by aphidicolin or SJK 132-20 at three points within S phase. They found that the total activity of pol ~t and pol 8 did not change as the cells progressed through S phase, but the ratio of pol 8 to pol ~ activity increased significantly. That observation suggested coordinate regulation of the two enzymes, and thus a function for each during S phase.

2.3. ROLE OF D N A POLYMERASEDELTA IN D N A REPLICATION

Although pol 8 has been known for about thirteen years (Byrnes el al., 1976), there is no direct evidence for its involvement in DNA replication. There are no known conditional mutants of pol 8, and, until recently, there were neither any selective small molecule inhibitors of pol 8 (Talanian et aL, 1989), nor any inhibitory monoclonal antibodies specific for pol 8 (Lee et al., 1989). In the absence of pol 8-specific inhibitors, the function of pol & has been inferred indirectly by measurement of the amount of replicative DNA synthesis which is resistant to inhibition by selective pol ct inhibitors but sensitive to inhibition by aphidicolin. Dresler and Frattini (1986, 1988) found that N L ( p - n - b u t y l p h e n y l ) - 2 ' - d e oxyguanosine 5'-triphosphate (BuPdGTP) (Wright and Dudycz, 1984), a potent inhibitor of pol ct (Wright and Dudycz, 1984; Khan et al., 1984) which inhibits pol 8 only weakly (Byrnes, 1985; Lee et al., 1985), inhibited replicative DNA synthesis in permeabilized human fibroblasts with a potency several orders of magnitude less than that which is displayed for pol ct. Dresler and Frattini concluded that the resistance of DNA synthesis to inhibition by BuPdGTP was due to the participation of a BuPdGTP-insensitive enzyme, possibly pol 8, in DNA synthesis. Hammond et aL (1987) made somewhat different observations but came to the same conclusion. They found that pol or-specific neutralizing monoclonal antibodies or BuPdGTP were capable of inhibiting replicative DNA synthesis in permeabilized CV-1 cells by roughly 50%, and that the remaining activity was sensitive to inhibition by aphidicolin. The latter results were consistent with the model that replicative DNA synthesis was catalyzed in equal parts by pol ct and pol 8. In similar experiments Basnakian et al. (1989) found that 80% of the

3. PROPERTIES OF DNA POLYMERASE DELTA 3.1. DNA POLYMERASEDELTA:A MAMMALIANDNA POLYMERASE WITH 3' --¢ 5' EXONUCLEASEACTIVITY

Pol 8 was first isolated from rabbit erythroid hyperplastic bone marrow (Byrnes et al., 1976). The enzyme has since been purified to different degrees from a variety of sources, including calf thymus (Lee et al., 1980), CV-1 cells (Hammond et al., 1987), rat heart (Zhang and Lee, 1987), human placenta (Lee and Toomey, 1987), rat neurons (Spadari et al., 1988) and HeLa cells (Nishida et al., 1988), suggesting that poi 8 is a ubiquitous mammalian cell DNA polymerase. The major feature of pol 8 which distinguished it most clearly from the other known mammalian DNA polymerases was its possession of a 3'--* 5' exonuclease activity, which could not be separated from, and thus seemed to be on the same peptide as, the DNA polymerase activity (Byrnes et al., 1976; Goscin and Byrnes, 1982). The 3'--*5' exonuclease activity of pol 8 could be inhibited by nucleoside 5'-monophosphates with little effect on the DNA polymerase activity (Byrnes et al., 1977), and, conversely, the DNA polymerase activity could be inhibited with little effect on the exonuclease activity (Talanian et al., 1989); these results suggest that, whether the active sites of the two activities are on the same peptide or not, they are structurally and functionally distinct. Most replication-specific prokaryotic DNA polymerases have an associated 3 ' ~ 5' exonuclease activity which increases the replication fidelity of the DNA polymerase by a 'proofreading' or 'editing' mechanism (Goodman, 1988; Kunkel, 1988). With few exceptions (Chen et al., 1979; Ottiger and Hfibscher, 1984; Skarnes et al., 1986; Cotterill et al., 1987; Reyland et al., 1988), experiments indicate

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R.V. TALANIANand G. E. WRIGHT

that pol ct is devoid of a 3' ~ 5' exonuclease activity, but only in the case of the Drosophila enzyme is it apparent that the exonuclease and polymerase activities reside on the same peptide (Cotterill et al., 1987; Reyland et al., 1988). The absence of 3' --* 5' exonuclease activity is the likely explanation for the relatively low replication fidelity of pol ~ in vitro (Loeb and Kunkel, 1982). Other mechanisms, such as the actions of proteins ancillary to pol ~t, must compensate in vivo for the low replication fidelity of the enzyme. The latter hypothesis is supported by the observation that the e subunit of E. coli DNA polymerase III, which contains a 3' ~ 5' exonuclease activity (Scheuermann and Echols, 1984) increased the in vitro fidelity of calf thymus pol ~ by 7-fold (Perrino and Loeb, 1989). In the case of pol 6, replication fidelity is increased significantly by the action of its associated 3' ~ 5' exonuclease activity (Byrnes et al., 1977; Kunkel et al., 1987; Sabatino and Bambara, 1988). High fidelity is an attractive feature for a replicative DNA polymerase, and thus the 3' ~ 5' exonuclease activity of pol 6 adds weight to the argument that it is a replicative DNA polymerase in vivo. 3.2. ENZYMATIC PROPERTIES OF D N A POLYMERASE DELTA: COMPARISON WITH D N A POLYMERASE ALPHA

Pol a and pol 6 can be distinguished on the basis of differences in the enzymatic properties of their DNA polymerase activities, as well as by the presence of associated activities. Pol 6 is most active on synthetic template: primers such as poly(dA) : oligo(dT) and the alternating copolymer poly(dA-dT), although the pattern of template preferences of the enzyme is strongly a function of its source and the method of its purification (So and Downey, 1988). Pol ct prefers template:primers with a high primer density, although, like pol 6, the template preference of pol ct is a function of its method and source of purification (Kaguni and Lehman, 1988). Pol a and pol 6 can also be distinguished on the basis of their processivity, the number of nucleotides that, on average, the polymerase adds to a growing primer chain before the enzyme dissociates from the template:primer. It is usually observed that pol ~t is a nonprocessive (distributive) enzyme, adding on the order of 5 to 100 nucleotides to a growing primer chain before dissociation (Fry and Loeb, 1986). The processivity of PCNA-sensitive pol 6, in the presence of that protein, is at least two orders of magnitude greater than that of pol ~t (Downey et al., 1988; So and Downey, 1988). In the absence of PCNA, PCNA-sensitive pol 6 synthesizes DNA in a distributive manner (Tan et al., 1986; Prelich et al., 1987b). The PCNA-insensitive form of pol 6 is highly processive in the absence of PCNA (Focher et al., 1989). The difference in the observed processivities of pol ct and pol 6 in vitro may not reflect their processivities in vivo; under the appropriate conditions, which may or may not reflect in vivo conditions, highly processive synthesis by both enzymes is possible (Sabatino et al., 1988). Another characteristic which distinguishes pol ct from pol 6 is that the latter is capable of strand-displacement synthesis (Downey et al., 1988), whereas the former is not (Murakami et al., 1986; Downey et al., 1988). Finally, pol ct contains a tightly

associated DNA primase activity which catalyzes the synthesis of RNA primers, allowing the enzyme to use single stranded DNA as a template (Fry and Loeb, 1986). With one possible exception (Crute et al., 1986), pol 6 does not appear to contain a DNA primase activity, and is completely dependent on the primase activity of another enzyme in order to use single stranded DNA as a template (Gassman et al., 1988). 3.3. PCNA: A STIMULATORYCOFACTOR OF DNA POLYMERASE DELTA

PCNA is a cell cycle regulated nuclear protein whose rate of synthesis closely tracks the rate of DNA synthesis in proliferating cells throughout the cell cycle (Celis et al., 1987). PCNA is identical with a previously described cell cycle-regulated nuclear protein called cyclin (Mathews et al., 1984). PCNA is an accessory protein of one form of pol 6 (PCNAsensitive pol 6) (Tan et al., 1986; Bravo et al., 1987; Prelich et al., 1987b), capable of stimulating the activity of that enzyme more than two orders of magnitude (Tan et al., 1986), depending on the assay conditions. The observations of Tan et al. (1986) and Prelich et aL (1987b) suggest that PCNA stimulates pol 6 by increasing its activity as well as its processivity. Preparations of pol 6 which do not contain PCNA and are insensitive to PCNA stimulation have been described (Crute et al., 1986; Focher et al., 1988b, 1989; Syvaoja and Linn, 1989); calf thymus has been the source of both PCNA-sensitive (Lee et al., 1984) and -insensitive (Focher et al., 1989) pol 6. The relationship between PCNA-sensitive and -insensitive forms of pol 6 is not clear, although the results of tryptic peptide mapping experiments suggest, at a minimum, that there are significant differences between the two (Wong et al., 1989). 3.4. STRUCTURAL RELATIONSHIP BETWEEN DNA POLYMERASES ALPHA AND DELTA

During most of the history of pol 6, the structural relationship between it and pol ct was unknown. The hypothesis that pol 6 was a precursor to or breakdown product ofpol ~t, or perhaps a posttranslationally modified form of pol ~t, seemed at least as likely as the hypothesis that they were different gene products (Byrnes and Black, 1978). Cotterill et al. (1987) observed that separation of the polymerase subunit from the four peptide Drosophila pol ~t-primase complex revealed a cryptic 3 ' ~ 5' exonuclease activity; later, the same group (Reyland et al., 1988) found that the isolated polymerase subunit was, like pol 6, far less sensitive to inhibition by BuPdGTP than the intact pol ct-primase. Demonstration that one enzyme under different conditions could exhibit the properties of pol ~ or pol 6 supported the hypothesis that pol ct and pol 6 were structurally related. However, recent observations seem to have tilted the argument in favor of the hypothesis that pol ~t and pol 6 are the products of different genes. First, Lee and Toomey (1987) showed by Western blotting that a mouse heteroantiserum raised against pol 6 reacted with pol 6 but not with pol ct, and that monoclonal antibodies raised against pol ~t displayed no reactivity with pol 6. The same group (Lee et al., 1989) later showed that

DNA polymerases alpha and delta of eight pol 6-neutralizing monoclonal antibodies, only two were inhibitory to pol ct, indicating that the two proteins are distinct but may share common structural features. Second, Focher et al. 0989) and Wong et al. (1989) independently showed by tryptic peptide analysis that the DNA polymerase catalytic subunits of calf thymus pol ct and pol 6 were different.

//a"

5'

g Strand

3I

5'

3.5. DIFFERENTIAL INHIBITORS OF D N A POLYMERASES ALPHA AND DELTA

Pol ct and pol 6 display similar sensitivities to aphidicolin (Lee et al., 1981; Goscin and Byrnes, 1982) and sulfhydryl-reacting compounds such as N-ethylmaleimide (Byrnes, 1984). Those observations have been cited as evidence for the hypothesis that pol ~ was a form ofpol ~. Khan et al. (1984) and Lee et al. 0985) showed that BuPdGTP and its dATP analog, 2-(p-n-butylanilino)-2'-deoxyadenosine 5'triphosphate (BuAdATP) (Khan et al., 1985) displayed differential inhibition of pol ct and pol 6, inhibiting the former enzyme with several orders of magnitude greater potency than the latter. Byrnes (1985) also showed that the pol s-neutralizing antibody SJK 132-20 (Tanaka et al., 1982) had no effect on pol & at concentrations which were completely inhibitory to pol ct. Compounds which inhibit pol 6 selectively with respect to pol ct have only recently been described. The authors showed that carbonyldiphosphonate (COMDP), a pyrophosphate analog, inhibited PCNA-insensitive pol 3 from calf thymus with twenty times greater potency than that which it displayed for calf thymus pol ct (Talanian et al., 1989). Further, COMDP inhibited the two enzymes by different mechanisms; it inhibited PCNAindependent pol ~ competitively with dNTPs, and inhibited pol ct uncompetitively with dNTPs (Talanian et al., 1989). Lee et al. 0989) have recently described eight monoclonal antibodies which neutralize PCNA-sensitive pol ~t derived from human placenta, and six of those antibodies were without effect on human placental pol ~t. Differential inhibition of pol ~t and pol 6 by both small molecules and monocional antibodies has reinforced the hypothesis that the two enzymes are distinct gene products.

4. WHY M I G H T THERE BE TWO REPLICATIVE DNA POLYMERASES IN M A M M A L I A N CELLS? 4.1. ASYMMETRY AT THE REPLICATION FORK All known DNA polymerases catalyze DNA synthesis by adding dNMP monomers to a growing primer chain exclusively in the 5'--* 3' direction (Kornberg, 1980). As a result, there is a fundamental asymmetry at the replication fork, wherein the 'leading strand' is synthesized toward the replication fork in a continuous manner, and the 'lagging strand' is synthesized away from the fork from a number of short RNA primers in a discontinuous manner (Fig. 1). The demands on DNA polymerases synthesizing leading and lagging strands are therefore likely to be very different.

109

~

g Strand

5' 3'

FIG. 1. Model of the DNA replication fork, in which two daughter strands (right) are synthesized from a parental strand (left). The leading strand (bottom) is synthesized in a continuous manner toward the replication fork, and the lagging strand (top) is synthesized away from the replication fork, in a discontinuous manner. 4.2. HYPOTHESIS" D N A POLYMERASES ALPHA AND DELTA CATALYZE LAGGING AND LEADING STRAND SYNTHESIS, RESPECTIVELY

Based on the properties of pol ~t and pol 6, and on a consideration of the task they face at the DNA replication fork, a model has been proposed in which the two enzymes act coordinately at the DNA replication fork as an asymmetric dimer, the former catalyzing lagging strand synthesis and the latter catalyzing leading strand synthesis (Focher et al., 1988a; Downey et al., 1988). Continuous synthesis on the leading strand would be catalyzed most efficiently by an enzyme with high processivity, while discontinuous synthesis on the lagging strand would be facilitated by an enzyme with low processivity. Frequent primer synthesis would be necessary during discontinuous synthesis, while continuous synthesis would require priming only rarely. Thus, the presence of a primase activity tightly associated with pol ct and not with pol 3 makes the former enzyme a more suitable candidate for lagging strand synthesis. Finally, the ability to conduct strand displacement synthesis, a capability of pol 6 but not of pol ct, would be crucial to the function of a leading strand DNA polymerase if it is required to replicate through duplex regions of DNA. The capacity to conduct strand displacement synthesis might, however, be a disadvantage to a lagging strand DNA polymerase, because it would enable the enzyme to displace, and thus waste, freshly synthesized daughter strands. 4.3. EVIDENCE FOR THE ASYMMETRIC DIMER HYPOTHESIS

In addition to a consideration of the properties of the replication fork and of the in vitro properties of pol ct and pol 6, the asymmetric dimer hypothesis is supported by several observations. Zhang and Lee 0987) observed that the ratio ofpol ~tto pol 6 activity in neonatal rat heart was constant during maturation and terminal differentiation of heart cells; Spadari

I10

R.V. TALANIANand G. E. WRIGHT

et al. (1988) made similar observations using developing rat neurons. Focher et al. (1988a) found that the ratio of pol ~ to pol 6 activity in calf thymus was invariably I : 1, using eight different extraction procedures, and in three subcellular locations. The participation of pol 6 in leading but not lagging strand synthesis is suggested by the finding that in an in vitro SV40 replication system, in the absence of PCNA, DNA synthesis occurred only on the lagging strand (Prelich and Stillman, 1988).

4.4. PRECEDENT FOR THE ASYMMETRIC DIMER HYPOTHESIS IN PROKARYOTIC SYSTEMS

The asymmetric dimer hypothesis of Focher et al. (1988a) and Downey et al. (1988) is not without precedent. It was first proposed in 1980 (Sinha et al., 1980) to explain the properties of a reconstituted T4 bacteriophage DNA replication system. The authors found that when a large excess of template DNA molecules was used in a T4 DNA polymerase reaction of limited duration, the small percentage of templates used were completely replicated. That observation indicated that, upon completion of the synthesis of an Okazaki fragment on the lagging strand, the lagging strand DNA polymerase was retargetted to the replication fork on the same template molecule. The asymmetric dimer hypothesis was proposed as a mechanism for that retargetting. McHenry has proposed (Johanson and McHenry, 1984; McHenry and Johanson, 1984; McHenry, 1985) that the E. coli DNA polymerase III (pol III) holoenzyme dimer, first proposed by Kornberg (1982), was asymmetric, and that one half of the dimer catalyzed leading strand synthesis while the other catalyzed lagging strand synthesis. Maki et al. (1988) and McHenry (1988) have suggested a structural basis for the asymmetry of the holoenzyme dimer. Each postulated that one of the halves of the dimer contains the z subunit, and the other contains the ~ subunit. Both proteins are products of the E. coli d n a X gene (Mullin et al., 1983; Kodaira et al., 1983), and the two differ by the absence in 7 of the carboxyl-terminal fourth of r (Maki and Kornberg, 1988a). The z subunit was proposed to increase the processivity of the pol III hoioenzyme (McHenry, 1982; Fay et al., 1982; Maki and Kornberg, 1988b), possibly by virtue of the several basic residues in its carboxyl-terminal domain (Yin et al., 1986; Flower and McHenry, 1986) which may strengthen binding to DNA. Thus, the processivitity of the r-containing half of the putative pol III holoenzyme makes it a good candidate as a leading strand polymerase, while the y-containing half may serve as the lagging strand polymerase, in an asymmetric dimer. 4.5. PRECEDENT FOR THE REQUIREMENT OF TWO POLYMERASES IN YEAST D N A REPLICATION

Yeast has provided a uniquely useful model for mammalian cell DNA replication because of the similarities between the yeast and mammalian cell chromosomes and because of the relative ease with which yeast genes can be cloned, mutagenized, and transfected (Campbell, 1986). Three DNA polymerases have been identified in yeast: yeast DNA

polymerases I, II, and III. Several lines of evidence suggest that yeast poi I is analogous to mammalian cell pol ct, and that yeast pol III is analogous to mammalian cell PCNA-sensitive pol 3. There is significant sequence homology between the genes encoding yeast pol I and pol ct (Pizzagalli et al., 1988). Yeast pol I, like pol ~t, was sensitive to inhibition by BuPdGTP (Burgers and Bauer, 1988) and had an associated DNA primase activity (Plevani et al., 1984; Singh and Dumas, 1984). Yeast pol III, like pol 3, possessed a Y - , 5 ' exonuclease activity and was devoid of a DNA primase activity (Bauer et al., 1988). Yeast pol III was stimulated by the PCNAsensitive pol 6 cofactor PCNA, and by the yeast analog of PCNA (Bauer and Burgers, 1988a; Burgers, 1988); the latter also stimulated PCNA-sensitive pol 6 (Bauer and Burgers, 1988b). Yeast pol III was also significantly less sensitive to inhibition by BuPdGTP than was yeast pol I (Burgers and Bauer, 1988). Yeast pol I was found to be required for replicative DNA synthesis (Budd and Campbell, 1987; Lucchini et al., 1988; Buddet al., 1989) and is encoded by a single copy, essential gene ( P O L l ) (Johnson et al., 1985). Yeast pol III is encoded by the C D C 2 gene (Sitney et al., 1989; Boulet et al., 1989), mutants of which have been found to be temperature-sensitive in DNA replication (Hartwell, 1967, 1976; Culotti and Hartwell, 1971). Thus, yeast sets the important precedent of an organism in which two distinct DNA polymerases are required for DNA replication; this precedent provides solid support for the asymmetric dimer hypothesis. 5. FUTURE PROSPECTS 5.1. ROLE OF DNA POLYMERASEDELTA IN MAMMALIAN

CELL DNA REPLICATION The hypothesis that pol 6 participates in mammalian cell DNA replication is supported only by limited circumstantial evidence and by the asymmetric dimer hypothesis, which is based on the in vitro properties of pol ~ and pol 6, and the properties of the replication fork. The function of pol 6 in vivo has been difficult to prove directly because of the absence of inhibitors specifically targetted to it, and because of the lack of conditional pol 6 mutants. While some progress has been made in inhibitor development, no pol 6 mutants have yet been described; the latter are potentially extremely informative. The inhibitory properties of COMDP, the first known small molecule which inhibits PCNA-insensitive pol 6 selectively with respect to pol ~t, have been described (Talanian et al., 1989). The ability of COMDP to inhibit PCNA-sensitive pol 6 has not been explored. If the compound inhibits PCNAsensitive pol 6 poorly compared to PCNA-insensitive pol 3, that observation will constitute further evidence that PCNA-sensitive and -insensitive pol 6 are distinct enzymes, and the compound will serve as a probe of the in vivo function of PCNA-insensitive pol 6 . If the compound inhibits the two forms of pol 6 with similar potencies, the results of in vivo studies will be ambiguous with respect to the contributions of the two pol 6 forms. The possibility also exists that COMDP will inhibit PCNA-sensitive pol 6

11l

DNA polymerases alpha and delta with significantly greater potency than it displays for PCNA-insensitive pol 3. That seems unlikely, because the Kt of COMDP [1.8 pM using poly(dA): oligo(dT) as the template:primer; Talanian et al., 1989] is within an order of magnitude of the most potent known inhibition of a DNA polymerase by a pyrophosphate analog (Oberg, 1989), suggesting that the binding of COMDP to PCNA-insensitive pol 6 is nearly optimal. The monoclonal antibodies described by Lee et al. (1989) also hold promise as in vivo probes of pol 3. The inhibitory properties of these antibodies toward PCNA-sensitive pol 3 have been described (Lee et al., 1989); it would be of great interest to measure their abilities to inhibit PCNAinsensitive pol 3. Measurement of the abilities of these antibodies to bind pol 6 derived from other sources, including yeast (yeast pol III), might quickly provide clues on the relationships between those enzymes. COMDP and monoclonal antibodies share a major shortcoming: because they are charged or large molecules, intact cell membranes are impermeable to them, and their use as probes of the function of pol 6 is limited to permeabilized cells, isolated nuclei, or reconstituted DNA replication systems. Identification of a small molecule inhibitor selective for pol 3 which can freely pass through the cell membrane, perhaps a nucleobase or nucleoside analog, would be of great value. 5.2. RELATIONSHIPBETWEENPCNA-SENSITIVEAND -INSENSITIVEDNA POLYMERASEDELTA The relationship between PCNA-sensitive and -insensitive pol 6 is not clear, but the available evidence suggests that they are distinct enzymes with different functions in the cell. The observations (discussed above) that SV40 (Prelich et al., 1987a) and CHO cell (Liu et al., 1989) DNA replication was dependent on PCNA and that PCNA-insensitive pol 3 was required for u.v. repair synthesis in a reconstituted system (Nishida et al., 1988; Syvaoja and Linn, 1989) suggest that PCNA-sensitive pol 3 is a replicative DNA polymerase, and that PCNA-insensitive pol 8 is a repair DNA polymeras¢, in mammalian cells. Although the structural relationship between the two forms of pol 3 is not known, the observation that tryptic peptide maps of the two enzymes derived from different sources showed little similarity (Wong et al., 1989) is consistent with the possibility that the two are products of separate genes. 5.3. RELATIONSHIPBETWEENDNA POLYMERASES ALPHA AND DELTA Although a large body of evidence suggests that pol ~t and pol 3 are different gene products, and that one is not merely an altered form of the other, definitive proof of that hypothesis is still lacking. The gene encoding human pol ~t has been cloned and sequenced (Wong et al., 1988). When the gene(s) encoding PCNA-sensitive and -insensitive pol 3 have also been cloned and sequenced, and their relationship to that of pol • defined, a clear statement on the structural relationship(s) between the two (or three) enzymes will be possible. The cloning of pol 3 will also allow in vivo probing of the mRNA encoding it, resulting

in informative biological studies which are not currently possible. Acknowledgements--The authors thank Dr Neal Brown for his critical review of the manuscript. Work in the authors' laboratory was supported by National Institutes of Health grant GM21747 (to G. E. W.).

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ZMUDZKA, B. Z., FORANCE, A., COLLINS, J. and WILSON, S. H. (1988) Characterization of DNA polymerase fl mRNA: Cell-cycle and growth response in cultured human cells. Nucleic Acids Res. 16: 9587-9596.

The roles of DNA polymerases alpha and delta in DNA replication.

The identities and precise roles of the DNA polymerase(s) involved in mammalian cell DNA replication are uncertain. Circumstantial evidence suggests t...
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