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Mutation Research, 236 (1990) 289-300 DNA Repair Elsevier MUTDNA 06012

Animal cell DNA polymerases in DNA repair F r e d W. P e r r i n o * a n d L a w r e n c e A. L o e b The Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, SM-30, University of Washington, Seattle, WA 98195 (U.S.~.~ (Accepted 12 March 1990)

Keywords: DNA polymerases; Exonucleases; Repair synthesis; Fidelity

The repair of DNA damage is a sequential multienzyme process. At a minimum, the damaged nucleotide(s) must be excised, the nucleotide sequence restored, and the newly synthesized DNA joined to adjacent nucleotides (Friedberg, 1985). Since the resynthesis of the nucleotide sequence after DNA damage is catalyzed by DNA polymerases, these enzymes must play a central role in DNA repair. It is the purpose of this review to appraise the roles of the different animal DNA polymerases in DNA repair and to present recent studies on the fidelity of DNA polymerases that relate to the accuracy of DNA repair. Damage to DNA occurs by both exogenous environmental agents and endogenous reactive cellular metabolites. The resultant lesions that are repaired include altered nucleotide bases, inter- or intra-strand DNA crosslinks, single- or doublestrand DNA breaks, as well as mismatched nucleotides resulting from deamination of 5-methylcytosine to thymidine or produced by misincorpotation during DNA synthesis. In prokaryotes, enzymes have been identified that recognize and

excise specific lesions in DNA. The involvement of different DNA polymerases in the resynthesis of DNA has been established by analysis of mutant enzymes, and DNA ligases rejoin the repaired DNA onto the contiguous DNA chain (Friedberg, 1985). Which DNA polymerase, or perhaps more appropriately, which DNA polymerase assembly functions in the various repair pathways in prokaryotes and eukaryotes is only beginning to be established. We will first outline the 4 classes of animal cell DNA polymerases in the following order a, ~, fl and "/ and then present some of the evidence that supports their involvement in genomic DNA repair. Secondly, we will consider the accuracy of restoration of the nucleotide sequence in DNA by analyzing the fidelity of the DNA polymerases in vitro. Lastly, we will analyze future research directions including the search for mutant DNA polymerases in animal cells.

Animal cell DNA polymerases D N A polymerase a

* Present address: Department of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, WinstonSalem, NC 27103 (U.S.A.). Correspondence: Dr. Lawrence A. Loeb, The Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, SM-30, University of Washington, Seattle, WA 98195 (U.S.A.), Phone 206-543-6015.

The major DNA polymerase activity in animal cells, DNA pol a, has been classically associated with DNA replication (Bollum, 1960). However, recent evidence suggests that this enzyme also has a major involvement in DNA repair. DNA pol a has been purified from a variety of animal cells by conventional protein separation methods or by immunoaffinity with monoclonal antibodies (Ta-

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290 naka et al., 1982; Wahl et al., 1984; Chang et al., 1984; Wang et al., 1984; Fry and Loeb, 1986). Determination of the molecular size of the DNA pol a catalytic polypeptide has been difficult due to the heterogeneity of catalytically active polypeptides produced during its purification. The spectrum of active fragments found in the most purified fractions has been attributed to DNA pol a's extreme susceptibility to proteolytic degradation. The use of protease inhibitors during immunoaffinity and rapid conventional chromatography diminishes in vitro proteolysis and results in the isolation of DNA pol ct activity that is associated with a polypeptide of molecular size between 140 and 180 kDa. Sequence analysis of the human DNA pol a gene further indicates that DNA pol a activity resides in the high molecular weight polypeptide (Wong et al., 1988). Proteolysis might not be solely an artifact of purification but might also occur physiologically. The active proteolyzed fragments could have different roles in cellular DNA metabolism. Various assemblies of DNA pol a have also been described. Enzymatic activities associated with the different forms of DNA pol a include primase, DNAases, C 1 and C2 primer recognition proteins, RNAase H, ATPase and diadenosine 5',5 '"-P1, p4_tetraphosphat e (Ap4A) binding activities (Loeb et al., 1986), and these activities have been considered to function in DNA replication. It remains to be determined which if any of these associated activities are involved in DNA repair. DNA polymerase 8 With the establishment of DNA pol 8 as an entity having many properties similar to DNA pol a, its function in DNA repair should also be considered. DNA pol 8 has been isolated from rabbit bone marrow, calf thymus, human placenta and HeLa cells using different conventional protein separation procedures (Byrnes et al. 1976; Byrnes, 1984; Lee et al., 1980, 1984; Lee and Toomey, 1987; Syvaoja and Linn, 1989). Polypeptides, presumed to be the catalytic subunit of DNA pol 8, have been identified in sizes ranging from 122 to 250 kDa (Bambara et al., 1989). A hallmark for DNA pol 8 is a tightly associated 3 ' ~ 5' exonuclease activity that is believed to

reside in the same polypeptide as the polymerase activity (Goscin and Byrnes, 1982). However, this association might not be diagnostic since the Drosophila DNA pol a contains a cryptic 3' ~ 5' exonuclease (Cotterill et al., 1987; Reyland et al., 1988). In one case, DNA pol 8 was purified in association with primase (Crute et al., 1986). A cell cycle dependent protein, PCNA (proliferating cell nuclear antigen, also called cyclin) has been shown to stimulate in vitro DNA synthesis by one form of DNA pol 8. Stimulation is manifested by an increase processivity when copying long single-stranded DNA stretches (Tan et al., 1986). Other forms of DNA pol 8 are affected less dramatically or not at all by PCNA (Focher et al., 1988; Bambara et al., 1989; Syvaoja and Linn, 1989). The presence of a 3' ~ 5' exonuclease associated with DNA pol 8 suggested that this polymerase would be more accurate than DNA pol a, and this has proven to be the situation (Kunkel et al., 1987). DNA polymerase fl DNA pol fl has long been considered the enzyme responsible for resynthesis of DNA after excision of damaged sites. It is the smallest and simplest of the eukaryotic DNA polymerases (Weisbach et al., 1971; Baril et al., 1971; Chang and Bollum, 1971). DNA pol fl is active as a single polypeptide of 39 kDa and is devoid of any known additional catalytic activities. The genes for both rat and human DNA pol fl have been cloned and their sequences determined (Zmudzka, et al., 1986; SenGupta et al., 1986). Milligram amounts of rat DNA pol fl have been purified from clones in E. coli (Abbotts et al., 1988). When purified from either Novikoff hepatoma or HeLa cells, DNA pol fl copurifies with, but is separable from, a bidirectional 3' -~ 5', 5' ~ 3' exonuclease DNAase V (Mosbaugh and Meyer, 1980; Mosbaugh and Linn, 1983). The association of DNA pol fl with a DNA-repair complex remains to be established. DNA polymerase y DNA pol y is usually purified from isolated mitochondria and is therefore presumed to be the mitochondrial replicative DNA polymerase. Early preparations of mitochondrial polymerase were

291 devoid of associated exonuclease activities. However, more recent studies show that a 3'--, 5' exonuclease copurifies with DNA polymerase activity (Kunkel and Mosbaugh, 1989). The molecular size of DNA polymerase -/, its subunit structure, and the identity of polypeptides that contain polymerase and exonuclease activities are uncertain. Early studies indicate that mitochondria fail to repair damaged DNA (Clayton et al., 1974). Although a role for DNA pol ~, in genomic DNA repair can not be eliminated, evidence for this is minimal and will not be considered here.

Repair of damaged DNA The most common mechanism for restoration of nucleotide sequence to damaged DNA is excision repair, a process involving excision of altered nucleotides and resynthesis of DNA. Time-course studies indicate that removal of DNA adducts (altered nucleotides) during DNA repair is biphasic. An initial rapid phase followed by a much slower phase has been observed in tissues from animals and in cultured cells after exposure to DNA-damaging agents (Bohr et al., 1987). The inherent complexities of DNA repair and the various methods for quantitating repair in animal cells has led to different interpretations about the nature of the biphasic removal of DNA adducts. One hypothesis predicts two pathways of DNA repair in animal cells (Regan and Setlow, 1974). An alternative explanation for the biphasic removal of DNA adducts is differential access to the damaged sites by DNA-repair enzymes due to chromatin structure (Lan and Smerdon, 1985) or active gene transcription (Mellon et al., 1986). Two repair pathways have been delineated based on the number of nucleotides incorporated during resynthesis of DNA. In 'long' patch repair approx. 100 nucleotides are incorporated per repaired segment: in 'short' patch repair only 3 or 4 nucleotides are incorporated (Friedberg, 1985). However, it must be emphasized that the pathways for repair of DNA and the number of nucleotides synthesized in a repair patch in animal cells remains controversial (Hanawalt et al., 1979).

Selective inhibitors of DNA polymerases In prokaryotes, mutations in genes coding for specific enzymes have provided the strongest evi-

dence for the function of that enzyme in cellular metabolism. Since few mutants of eukaryotic DNA polymerases are available, other less exacting methods have been used, among which are selective DNA polymerase inhibitors. Typically, the effect of the inhibitor is first determined in vitro using different purified DNA polymerases. DNA damage is then induced in either whole or permeable cells and the inhibition of DNA repair is studied using concentrations of the inhibitor that allows one to infer which DNA polymerase might function in the repair process. Aphidicolin, an antibiotic isolated from the fungus Cephalosporium aphidicola, inhibits in vitro DNA synthesis by both DNA pols a and 8 at concentrations (1-40 /~g/ml) that do not inhibit the activity of DNA pol fl (Ikegami et al., 1978; Spadari et al., 1984; Huberman, 1981; Goscin and Byrnes, 1982). In contrast, the 2',3'-dideoxynucleoside-5'-triphosphates (ddNTPs), analogs of the usual substrates for DNA polymerases 2'-deoxynucleoside-5'-triphosphates, that lack the 3'oxygen in the deoxyribose moiety are inhibitors of DNA pol ft. DNA pol fl activity is more sensitive to these analogs than is DNA pol 8 which is more sensitive than DNA pol a (Waqar et al., 1984; Wahl et al., 1986). Distinguishing between D N A pols a and 8 has been difficult due to their similar inhibitor sensitivities. However, butylphenyldGTP and butylanilinodATP have been used to distinguish between these DNA polymerases (Wright and Dudycz, 1984). DNA pol a is inhibited by concentrations of these analogs of about 1 ~M whereas DNA pol 8 is inhibited only at concentrations of 100/~M (Kahn et al., 1984; Byrnes, 1985; Wahl et al., 1986). Certainly, one must interpret cautiously the correlative observations of inhibitor effects on pure enzymes relative to effects in crude systems such as whole or permeable cells. Nevertheless, selective inhibition analysis can be a valuable step towards the assignment of in vivo roles for DNA polymerases.

Aphidicofin-sensitivity of DNA polymerases a and 8 The ability of aphidicolin to inhibit DNA-repair synthesis has been studied using cells exposed to a variety of agents that damage DNA. Exposure of cells to UV-irradiation induces two major photoproducts, pyrimidine-pyrimidine and

292 pyrimidine-pyrimidone dimers (Basu and Essigmann, 1988). The repair of these photoproducts is thought to occur predominately via a 'long' patch pathway (Hanawalt et al., 1979). Repair synthesis, detected in UV-irradiated HeLa cells under conditions that minimized replicative DNA synthesis, is effectively inhibited by aphidicolin (Hanaoka et al., 1979). These observations suggest that an aphidicolin-sensitive DNA polymerase is involved in repair of UV-induced damage, and is responsible at least in part for 'long' patch repair. The participation of an aphidicolin-sensitive DNA polymerase in UV-induced DNA repair has been further substantiated using permeable human lymphoblasts (Berger et al., 1979), as well as osmotically lysed (Ciarrocchi et al., 1979) and confluent cultures of human fibroblasts (Snyder and Regan, 1981; Collins, 1983). Furthermore, it has been shown that repair synthesis induced by a variety of other DNA-damaging agents is also aphidicolin-sensitive; these include: X-irradiation, methyl methanesulfonate, dimethylnitrosoamine, N-methyl-N'-nitro-N-nitrosoguanidine, N-nitrosomethylurea, and N-methyl-N-nitrosourea (Fry and Loeb, 1986). Increasing doses of the damaging agents, presumably producing a parallel increase in DNA damage, require progressively higher levels of aphidicolin-sensitive DNA polymerase (Dresler and Lieberman, 1983). Until recently, DNA polymerase a was thought to be the only animal DNA polymerase sensitive to inhibition by aphidicolin. Thus, inhibition of repair synthesis by this drug was equated with inhibition of DNA pol a. However, DNA pol 8, an enzyme with many properties similar to DNA pol a, is also inhibited by aphidicolin. To distinguish between these two enzymes, investigators used butylphenyldGTP, a strong inhibitor of in vitro DNA synthesis by DNA pol a and a weak inhibitor of DNA pol 8. Using this nucleotide analog, Dresler and colleagues (1986, 1988) measured inhibition of DNA-repair synthesis induced by UV-irradiation in permeable human fibroblasts. They concluded that both the early and late phases of DNA repair were mediated by DNA pol 8, rather than DNA pol a. A role for DNA pol 8 in repair of UV damage is also supported by the determination that a depleted repair factor in permeable human fibroblasts can be complemented

with purified DNA pol 8 from HeLa cells (Nishida et al., 1988). DNA pol 6 was further implicated by correlating the inhibition of DNA-repair synthesis using ddTrP with the relative sensitivities of DNA pols a, /3 and 8 with this analog (Dresler and Kimbro, 1987). The above results assume that the sensitivities of DNA polymerases to inhibitors are the same for the purified enzyme as in crude systems. Furthermore, it remains to be determined if DNA pols a and 8 are truly the products of different genes or simply different biochemical forms of the same DNA polymerase.

Aphidicolin-resistance of DNA polymerase fl The repair of DNA damage induced by ionizing radiation, bleomycin and some alkylating agents is thought to be mediated through a 'short' patch repair pathway (Hanawalt et al., 1979). Studies with animal cells using selective DNA polymerase inhibitors suggest that DNA pol fl participates in this pathway. When HeLa cells are damaged with dimethyl sulfate, neocarzinostatin or bleomycin, the reduced ability to repair the damage in the presence of ddTTP or aphidicolin indicates the participation of both DNA pols fl and a (Yamada et al., 1985). The amount of DNA damage could determine the utilization of a particular DNA polymerase in repair. Low amounts of DNA damage have been proposed as the substrate for repair by DNA pol fl (Dresler and Lieberman, 1983). Other studies indicate the nature of the DNA damage determines which DNA polymerase is employed (Miller and Chinault, 1982a). For example, repair synthesis induced by bleomycin damage appears to be ddTTP-sensitive and thus might be mediated, in part, by DNA pol /3 (Seki et al., 1982; Miller and Chinault, 1982b). In contrast, repair synthesis that is sensitive to aphidicolin such as the repair of UV-photoproducts might be mediated by DNA pols a or 6. In prokaryotes, DNA damage is frequently accompanied by the induction of enzymes required for the repair of that damage. However, inducible DNA repair has not been unequivocally demonstrated in eukaryotes. In accord with the role of DNA pol fl in DNA repair it has been shown that mRNA for DNA pol /3 varies only slightly during the cell cycle and is similar in non-dividing and dividing cells (Zmudzka et al.,

293

1988). After treatment of CHO cells with certain DNA-damaging agents the m R N A levels increase, but this increase in m R N A does not correlate with a parallel increase in the DNA pol fl protein (Fornace et al., 1989). These results suggest some form of control for DNA pol fl in DNA repair at the level of translation. In vitro repair assays Reconstructed in vitro repair systems have been designed to assess the potential of DNA pol a and fl in well defined modes of DNA repair. An in vitro repair system reconstituted with purified enzymes from HeLa cells has been used to implicate DNA pol fl in 'short' patch repair (Mosbaugh and Linn, 1983). Evans and Linn (1984) have extended this in vitro system to the repair of UV-induced damage in SV40 minichromosomes implying that DNA pol fl is involved in repair of UV-induced damage. However, this does not exclude a role for DNA pol a, since in a model system 'short' patch repair of depurinated DNA can be carried out by DNA pol a (Bose et al., 1978). In another situation, the specific correction of G : T mispairs to G : C pairs has been demonstrated in vitro with HeLa cell extracts (Brown and Jiricny, 1987; Wiebauer and Jiricny, 1989). DNA pol fl was implicated for resynthesis in this system by incorporation of ddNTPs and by its inhibition using antibodies against DNA pol fl (Jiricny, unpublished results). Recently, a cell-free repair assay using a chemically synthesized abasic site has been developed (Matsumoto and Bogenhagen, 1989). All of the sequential reactions from excision to ligation were demonstrated. Identification of the DNA polymerase responsible for resynthesis in this specific repair system should be forthcoming.

Fidelity of DNA polymerases in vitro Fidelity of DNA synthesis has been mainly considered with respect to DNA replication. With the designation of different DNA polymerases as repair enzymes it is instructive to consider measurements on the fidelity of DNA polymerases within the context of DNA repair. In the case of DNA replication, measurements of spontaneous mutation rates provide a convenient yardstick to

gauge the accuracy of the overall DNA-replication process. Since the spontaneous mutation rate in eukaryotic cells is about 10-1°-10 -12 error per nucleotide synthesized per cell division (Drake et al., 1969), it seems likely that DNA replication must be at least as accurate. In the case of DNArepair synthesis, we lack measurements on nucleotide misincorporation within repair patches, and it is thus conceivable that DNA repair might be highly error prone. Fidelity of DNA polymerase a in vitro Our studies on the fidelity of DNA pol et have been considered mostly within the context that DNA pol a is the replicative enzyme. However, our in vitro results might just as closely mimic the DNA synthetic process that is likely to occur during DNA repair, i.e. copying stretches of single-stranded DNA templates such as those perhaps produced as a consequence of DNA damage. The catalytic steps of DNA synthesis in vitro can be visualized as a series of alternative pathways in which discrete decisions by DNA polymerase are exercised during each nucleotide addition step (Fig. 1). For correct incorporation of the next nucleotide (insertion), a DNA polymerase binds to the 3' terminus, 'reads' the template base, selects the complementary nueleotide, positions the substrate nueleotide opposite the template nucleotide, and catalyzes its covalent linkage onto the growing 3' terminus. Two pathways must be considered after the D N A polymerase misinserts a nucleotide (misinsertion). The DNA polymerase can either incorporate the next complementary nucleotide onto the mispaired 3' terminus thus fixing the mismatch into duplex DNA (mispair

5'

3'

3'

AT

Insertion D

TA AT

I

Mispair Extension

Misinsertion

AT 5'

/A

J Proofreading by 3'~5' Exonuclease

__AT.__

AT

Fig. 1. Alternative pathways for DNA polymerase at each nucleotide addition step.

294

extension) or it can terminate synthesis providing an opportunity for a 3'--> 5' exonuclease to remove the terminal mispair (proofreading). DNA pol a is usually purified devoid of 3 ' ~ 5' exonuclease activity; however, exonucleolytic proofreading in vivo might be provided by a separate polypeptide that is lost during purification. In the absence of an exonuclease, the primary determinants of fidelity of D N A pol a in vitro are the frequency of misincorporation and the efficiency of mispair extension. At the insertion step (Fig. 1), DNA pol a discriminates between correct and incorrect nucleotides; the frequency for misinsertion of an incorrect nucleotide is about 1 / 2 0 000 (Boosalis et al., 1987; Perrino and Loeb, 1989a). At the extension step, D N A pol a extends the resultant mispaired 3' termini with low efficiency (Perrino and Loeb, 1989a). These two steps can be demonstrated by displaying newly elongated D N A primers after polyacrylamide gel electrophoresis. When singly-primed ~/iX174 am3 D N A is copied by calf-thymus D N A pol a with equal concentrations of all 4 nucleotides no pausing by D N A pol a is detected within several hundred nucleotides of the amber3 site. With progressive reduction of dTTP concentrations in the reaction, pause sites become visible opposite adenine residues in the template strand (Fig. 2). The location of these pause sites corresponds to nucleotide additions opposite the template adenines and presumably represents extended primers with a misincorporated base at the 3' terminus. This low efficiency of mispair extension contrasts with other polymerases lacking 3' ~ 5' exonucleases that we have tested including rat D N A polymerase-fl, AMV polymerase and E. coli DNA polymerase III-ct subunit; with these polymerases the major pause sites with decreased dTTP occur prior to and not opposite template A's (Fig. 3). The finding that D N A pol a extends mispairs inefficiently suggests that a separate 3 ' ~ 5' exonuclease might exist in mammalian cells to excise nucleotides misinserted by DNA pol a. We tested this hypothesis by determining whether the c subunit of E. coil D N A polymerase III could enhance the accuracy of D N A pol a in vitro and thus substitute for a putative proofreading component that might be present as a separate polypeptide in

A _____1~ A -----~ A---~ o

(~X 174 am3 Template A----~

T A---~ GT

A~

TG C G C T G C T T T G T G G

Primer---~

#JZ! LLL.c._, dTTP (tiM) Fig. 2. Pause sites by calf-thymus D N A pol a resulting from nucleotide biasing. ~ X 1 7 4 am3 D N A was primed with 5'32p-labeled 15-mer and copied with D N A pol a in reactions containing 1 m M dATP, dCTP, dG TP and the indicated concentration of d'l-'rP. The control is a reaction to which D N A pol a but no nucleotides were added. Pausing opposite the template adenines and the primer position are indicated. For details see Perrino and Loeb (1989a).

animal cells. The ~ subunit of E. coli DNA polymerase III is the 3' ---, 5' exonuclease that removes nucleotides misinserted by D N A pol III during replication (Scheuermann and Echols, 1984).

295

i ilii!iliii!iiliiii !!i!ii!ilgiil ! ! ! ! ! i i i l i

A

i~iiii~i!i!i!i!iii~!ilLi~!!i!~ii~i~ii!~i

A A

i!ii!iii~i~iiiiii!i~ii~i~iiiiii~i

~)X 174 am3 Template -sequence

ili]!iii!,i ¸i A

PrJmer---I~

~ii

ii;iiliili

Primer

i ..........

JJ JJ I,LL O 5 15 30

C.T. pol C~-primase

AMV Pol ]]IG-5ubunit

Fig. 3. Pause sites by D N A polymerases lacking 3' ---,5' exonucleases resulting from nucleotide biasing. ~ X 1 7 4 am3 D N A was primed with 5'-32p-labeled 15-mer and copied with equal activities of the indicated D N A polymerase in reactions containing 1 m M dATP, dCTP, d G T P and 1 / t M dTTP. Positions of the template adenines and primer are indicated. For details see Perrino and Loeb (1989a).

Nucleotide misinsertions by DNA pol a result in! 3' terminal mispairs which impeded further DNA synthesis (Fig. 4). The c subunit hydrolyzes these mispairs and permits further elongation by DNA

Time (rain) Pol-cz only

5 15 3 0

+ ~- subunit and dTTP

Fig. 4. The 3' terminal mispairs generated by calf-thymus D N A pol a are hydrolyzed by the ~ subunit of E. coli D N A polymerase III. ~ X 1 7 4 am3 D N A was primed with 5'-32p labeled 15-mer and copied with D N A pol a in a reaction containing 1 m M dATP, d C T P and dGTP. Samples were removed after D N A pol et addition (time 0) and at the indicated times after incubation. At 30 min, c subunit and dTTP were added and samples were removed at the indicated times. The positions of the primer and template adenines are indicated. For details see Perrino and Loeb (1989b).

pol a. The cooperation between these two enzymes suggests that a separate 3' --* 5' proofreading exonuclease could functionally interact with

296 DNA pol a to achieve highly accurate DNA synthesis. It remains to be determined whether accessibility to the 3' terminal mispair by the exonuclease is facilitated by dissociation of DNA pol a. It is possible that D N A pol a remains associated with the 3' terminus and changes conformation to position the terminal mispair so that it is accessible to hydrolysis by the separate 3' ~ 5' exonuclease. On the basis of these observations, we designed a model in vitro proofreading assay that exploits D N A pol a's extremely slow rate of extension from a 3' terminal A: G mispair. A template: primer was constructed by hybridizing an oligonucleotide primer of 16 nucleotides to a template of 30 nucleotides. 15 of the 16 nucleotides in the primer are complementary to the template, and the 3' terminus forms an A : G mismatch. We observed that the ~ subunit of DNA polymerase III removes the terminal mispair and that D N A pol a extends the newly corrected 3' terminus. We proposed that a similar 3' --* 5' proofreading exonuclease activity might be present in mammalian cells and could be identified using this assay. Fidelity of DNA polymerase 8 in vitro DNA pol 8 is highly accurate during in vitro D N A synthesis, producing less than one error per 10 6 nucleotides polymerized (Kunkel et al., 1987). The 3 ' ~ 5' exonuclease activity associated with D N A pol 8 is instrumental in establishing this high accuracy. D N A pol 8's exonuclease preferentially excises non-complementary nucleotides from the 3' terminus (Sabatino and Bambara, 1988), and inhibition of this exonuclease results in increased misincorporation by the DNA polymerase (Kunkel et al., 1987). DNA polymerases a and 8 in vitro Recent in vitro studies indicate that DNA pols a and 8 might act coordinately during bidirectional semiconservative replication of SV40 D N A (Kelly, 1988; Decker et al., 1987; Prelich and Stillman, 1988; Lee et al., 1989). It has been hypothesized that DNA pol a is responsible for lagging strand synthesis and D N A pol 8 for leading strand synthesis. Initial reports using soluble extracts, presumably containing both DNA pols a and 8, suggest that DNA synthesis in the SV40

system is highly accurate (Roberts et al., 1988). The mechanism for this high accuracy is not known. We have recently shown that the 3 ' ~ 5' exonuclease associated with DNA pol 8 removes terminal mispairs to produce correctly paired 3' termini that can be subsequently extended by D N A pol a (Perrino and Loeb, 1990). Thus, the exonuclease associated with DNA pol 8 can proofread for DNA pol a in vitro. These two D N A polymerases could act coordinately during both D N A replication and repair. Fidelity of DNA polymerase fl in vitro D N A pol /3 is more error-prone than DNA pols a and 8. The error rate for single-base substitutions is greater than 1/5000 (Loeb and Kunkel, 1982; Kunkel, 1985a). Base substitution errors during in vitro synthesis by D N A pol /3 can arise either directly from nucleotide misinsertion or indirectly as a consequence of transient misalignment of the 3' terminus with the template nucleotide (Boosalis et al., 1989). The fidelity of in vitro D N A synthesis by DNA pol /3 can be distinguished from that by D N A pol a by 3 criteria: DNA pol /3 (1) more readily produces errors by a 'transient dislocation' mechanism, (2) more efficiently extends terminal mispairs (Korn et al., 1983), and (3) produces a different spectrum of errors than DNA pol a (Kunkel, 1985b; Kunkel and Bebenek, 1989). Future directions

Like D N A replication, DNA repair could be mediated by multienzyme complexes (Lee and Sirover, 1989). Several approaches will likely be required to delineate the roles of the different D N A polymerases in the different pathways of D N A repair. If a spectrum of errors produced during DNA repair or replication could be established in vivo, the different errors produced by different D N A polymerases in vitro might indicate which D N A polymerase carried out the repair synthesis. Towards this end, studies on the spectrum of mutations in the aprt gene suggest that exonucleolytic proofreading occurs during DNA replication in vivo (Phear et al., 1988), thus implicating D N A pol 8 or a separate exonuclease that works concertedly with D N A pols a or ft.

297

Despite correlative studies in vivo and in vitro, an unambiguous assignment of a D N A polymerase to a DNA-repair pathway is likely to require studies with conditional mutants in D N A polymerases, and these are only now becoming available. The studies of mutant cells containing an aphidicolin-resistant D N A polymerase defective in D N A repair establish the involvement of either DNA pol et or fl in repair synthesis (Liu et al., 1983). However, it must now be determined which of these two enzymes (or both) is responsible for the aphidicolin resistance. It seems likely that a variety of mutant DNA polymerases will be identified on the basis of drug resistance or by screening for mutator or antimutator phenotypes. A number of human hereditary diseases result from defects in D N A repair, and thus cells from these individuals could contain altered D N A polymerases. Isolation of the different D N A polymerases from these cells might be instrumental in establishing this association. Complementation of mutations in E. coli or yeast by genes from animal cells might provide a genetic selection for D N A repair or replication enzymes. The complementation of a repair deficient mutant in E. coli with the Saccharomyces cerevisiae 3-MeA D N A glycosylase gene provides a prototype for this approach (Chen et al., 1989).

Acknowledgements We thank Kris Carroll for help in preparing the figures. This work was supported in part by N I H grants T32-CA-09437 (F.W.P.) and R35-CA-39903 (L.A.L.).

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