Yeast DNA Polymerase {zeta} Is an Efficient Extender of Primer Ends Opposite from 7,8-Dihydro-8-Oxoguanine and O6-Methylguanine

Genetic studies in Saccharomyces cerevisiae have indicated therequirement of DNA polymerase (Pol) ${zeta}$ for mutagenesis inducedby UV light and by other DNA damaging agents. However, on itsown, Pol ${zeta}$ is highly inefficient at replicating through DNA lesions;rather, it promotes their mutagenic bypass by extending fromthe nucleotide inserted opposite the lesion by another DNA polymerase.So far, such a role for Pol ${zeta}$ has been established for cyclobutanepyrimidine dimers, (6-4) dipyrimidine photoproducts, and abasicsites. Here, we examine whether Pol ${zeta}$ can replicate through the7,8-dihydro-8-oxoguanine (8-oxoG) and O⁶-methylguanine (m6G)lesions. We chose these two lesions for this study because thereplicative polymerase, Pol ${delta}$ , can replicate through them, albeitweakly. We found that Pol ${zeta}$ is very inefficient at inserting nucleotidesopposite both these lesions, but it can efficiently extend fromthe nucleotides inserted opposite them by Pol ${delta}$ . Also, the mostefficient bypass of 8-oxoG and m6G lesions occurs when Pol ${delta}$ iscombined with Pol ${zeta}$ , indicating a role for Pol ${zeta}$ in extending fromthe nucleotides inserted opposite these lesions by Pol ${delta}$ . Thus,Pol ${zeta}$ is a highly specialized polymerase that can proficientlyextend from the primer ends opposite DNA lesions, irrespectiveof their degree of geometric distortion. Pol ${zeta}$ , however, is unusuallysensitive to geometric distortion of the templating residue,as it is highly inefficient at incorporating nucleotides evenopposite the moderately distorting 8-oxoG and m6G lesions.

INTRODUCTION

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Introduction

DNA lesions often block the progression of the replication fork,but replication through such lesions can be achieved by theaction of specialized DNA polymerases. Eukaryotic cells containa multiplicity of such DNA polymerases (Pols), such as Pol ${eta}$ ,Pol ${zeta}$ , and others. Pol ${eta}$ is unique among eukaryotic polymerases(9) in its ability to replicate through a cis-syn thymine-thymine(TT) dimer efficiently and accurately (12, 20), and geneticstudies in yeast have indicated a role for Pol ${eta}$ in the replicationof cyclobutane pyrimidine dimers (CPDs) formed at TC and CCsites (22). Because of the requirement of Pol ${eta}$ for the error-freebypass of CPDs, its inactivation in humans (8, 14) causes anincrease in the incidence of UV mutagenesis (18, 21) and resultsin the cancer-prone syndrome, the variant form of xerodermapigmentosum. Pol ${eta}$ can also replicate through other DNA lesionssuch as 7,8-dihydro-8-oxoguanine (8-oxoG) and O⁶-methylguanine(m6G) (4, 6). The competency of Pol ${eta}$ to replicate through theseDNA lesions derives from its proficient ability to both insertnucleotides opposite the lesion site and to extend from theinserted nucleotide.

By contrast to the proficient ability of Pol ${eta}$ to replicate throughsome DNA lesions, replication through certain DNA lesions requiresthe sequential action of two DNA polymerases, in which one polymeraseinserts the nucleotide opposite the lesion site and the otherextends from the inserted nucleotide (for a review, see reference17). Genetic studies in the yeast Saccharomyces cerevisiae haveprovided evidence for the requirement of Pol ${zeta}$ for mutagenesisinduced by UV light and by other DNA damaging agents (10, 13),thus indicating a role for Pol ${zeta}$ in mutagenic translesion synthesis(TLS). But, on its own, Pol ${zeta}$ is unable to replicate through DNAlesions because it is highly inefficient at inserting nucleotidesopposite the lesion site. Pol ${zeta}$ , however, is very proficient atextending mismatched primer termini on undamaged DNAs and alsoin extending from nucleotides inserted opposite lesion sites(17). Thus, although Pol ${zeta}$ is very inefficient at inserting nucleotidesopposite the 3' T of a cis-syn TT dimer or a (6-4) TT photoproduct,it can proficiently extend from a G nucleotide inserted oppositethe 3' T of these lesions by another DNA polymerase (7, 11).Pol ${zeta}$ is also very inefficient at inserting nucleotides oppositean abasic site, but it proficiently extends from nucleotides,particularly an A, inserted opposite an abasic site by anotherDNA polymerase (5).

Oxygen-free radicals attack bases in DNA, and 8-oxoG is oneof the adducts formed. Treatment of DNA with an alkylating agentsuch as N-methyl-N'-nitro-N-nitrosoguanidine results in theformation of m6G in DNA. Yeast Pol ${eta}$ replicates through the 8-oxoGlesion by proficiently inserting a C and by proficiently extendingfrom this nucleotide (6). Pol ${eta}$ replicates through the m6G lesionby inserting a C or a T nucleotide opposite the lesion and thenextending from the inserted nucleotide (4). Although the replicativeyeast DNA polymerase, Pol ${delta}$ , is also able to incorporate nucleotidesopposite 8-oxoG and m6G lesions and to extend from the insertednucleotides, it is quite inefficient at both these steps (4,6). Nevertheless, because Pol ${delta}$ would be the first polymeraseto arrive at the lesion site, it could still carry out the insertionstep but then stall because of its inefficient ability to extendfrom the nucleotide inserted opposite the lesion. In that case,the completion of lesion bypass would necessitate the actionof an extender polymerase such as Pol ${zeta}$ . Here, we examine theability of yeast Pol ${zeta}$ to replicate through the 8-oxoG and m6Glesions. Using steady-state kinetic analyses, we show that Pol ${zeta}$ is highly inefficient at inserting nucleotides opposite boththese lesions, but it can efficiently extend from the nucleotideinserted opposite these lesions by Pol ${delta}$ . These results underscorethe highly specialized role of Pol ${zeta}$ at the extension step inlesion bypass.

MATERIALS AND METHODS

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Materials and Methods

Proteins. S. cerevisiae Pol ${zeta}$ was expressed in yeast strain Sc334as a glutathione S-transferase (GST)-Rev3 fusion protein incomplex with Rev7 protein as described previously (7). The GST-Rev3/Rev7-containingfractions were concentrated using Microcon 30 (Amicon) and frozenat -70°C. S. cerevisiae Pol ${delta}$ was kindly provided by PeterBurgers.

DNA substrates. Oligonucleotides were synthesized by Midland Certified ReagentCo. (Midland, Tex.). DNA S1 substrates, shown below in Fig.1, were generated by annealing a 75-nucleotide (nt) oligomertemplate, 5'-AGC TAC CAT GCC TGC CTC AAG AAT TCG TAA XAT GCCTAC ACT GGA GTA CCG GAG CAT CGT CGT GAC TGG GAA AAC-3', containinga G, an 8-oxoG, or an m6G at position 31 (X), to the 40-nt 5'-³²P-labeledoligonucleotide primer, N4264 (5'-GTT TTC CCA GTC ACG ACG ATGCTC CGG TAC TCC AGT GTA G-3'). For DNA S2 substrates, shownbelow in Fig. 2, and also used for steady-state kinetic analysisof extension reactions below in Fig. 3, the 75-nt oligomer templatesused for generating the S1 substrates were annealed to the 5'³²P-labeled oligonucleotide primers, 5'-GTT TTC CCA GTC ACGACG ATG CTC CGG TAC TCC AGT GTA GGCATN-3', where N is G, A,T, or C. For steady-state kinetic analysis of the insertionreactions shown below in Fig. 3, the 75-nt templates used forgenerating the S1 substrates were annealed to the 5'-³²P-labeledoligonucleotide primer, N4309 (5'-GTT TTC CCA GTC ACG ACG ATGCTC CGG TAC TCC AGT GTA GGC AT-3').

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FIG. 1. Bypass of 8-oxoG and m6G lesions by the combined action of Pol ${delta}$ and Pol ${zeta}$ . (A) DNA synthesis on an 8-oxoG-containing DNA template. Lanes 1 to 7, undamaged DNA; lanes 8 to 14, 8-oxoG-containing DNA. Sequences adjacent to the primer-template junction are shown for the 40-nt, 5'-³²P-labeled primer and 75-nt template. The position corresponding to the undamaged G or the 8-oxoG site on the template is indicated by _*G. Yeast Pol ${zeta}$ (10 nM), yeast Pol ${delta}$ (10 nM), or a combination of these two enzymes were incubated with the DNA substrate (20 nM) in the presence of each of the four dNTPs (5 or 100 µM) at 30°C for 10 min. The reaction products were resolved on a 15% denaturing polyacrylamide gel and visualized by autoradiography. The gel was analyzed by using a PhosphorImager, and the concentrations of the products of the synthesis past the undamaged G or 8-oxoG are indicated. (B) DNA synthesis on an m6G-containing DNA template. Lanes 1 to 7, undamaged DNA; lanes 8 to 14, m6G-containing DNA. Reactions were carried out as indicated in the legend for panel A, except that the incubation time of the reactions was 15 min.

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FIG. 2. Extension of primers with various 3' ends opposite 8-oxoG and m6G lesions by Pol ${zeta}$ . Each of the four different primers, differing only in the 3'-terminal nucleotide, was annealed to the template oligonucleotide containing an undamaged G, an 8-oxoG, or an m6G residue at the same position. In the primer-template pair shown, N designates the position of the variable terminal primer nucleotide and _*G designates the position of a G, an 8-oxoG, or an m6G residue. Pol ${zeta}$ (10 nM) was incubated with the DNA substrate (20 nM) in the presence of each of the four dNTPs (100 µM) at 30°C for 15 min. The gel was analyzed by using a PhosphorImager, and the amount of the product of primer extension opposite from the undamaged G, 8-oxoG, or m6G is indicated.

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FIG. 3. Steady-state kinetic analysis of insertion and extension reactions catalyzed by Pol ${zeta}$ on 8-oxoG- and m6G-containing DNA templates. A portion of the DNA substrate used for insertion reactions (top left) or for extension reactions (top right) is shown, and the position of an 8-oxoG, m6G, or undamaged G residue in the template oligonucleotide is indicated by an asterisk (top). Pol ${zeta}$ (2 nM) was incubated with the primer-template DNA substrate (30 nM) and increasing concentrations of a single deoxynucleotide for 10 min at 30°C. The quenched samples were analyzed by 15% denaturing polyacrylamide gel electrophoresis, and the steady-state kinetic parameters k_cat and K_m were determined.

DNA polymerase assays. A standard DNA polymerase reaction mixture (10 µl) contained25 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 5 mM dithiothreitol, 100µg of bovine serum albumin/ml, 20 nM 5'-³²P-labeled oligonucleotideprimer annealed to an oligonucleotide template, and 5 or 100µM concentrations of each of all four deoxynucleotides(dGTP, dATP, dTTP, and dCTP), as indicated in the figure legends.Reactions were started by the addition of Pol ${zeta}$ (10 nM), Pol ${delta}$ (10nM), or a mixture of Pol ${delta}$ and Pol ${zeta}$ and incubated at 30°C for10 to 15 min followed by quenching by the addition of loadingbuffer (40 µl) containing EDTA (20 mM), 95% formamide,0.3% bromphenol blue, and 0.3% cyanol blue. The reaction productswere resolved on 15% polyacrylamide gels containing 8 M urea.Quantitation of the results was done using a Molecular DynamicsSTORM PhosphorImager and ImageQuant software.

Analysis of steady-state kinetics. Steady-state kinetic analyses for deoxynucleotide incorporationopposite an 8-oxoG or an m6G, or primer extension opposite fromthese lesions were performed as described previously (2, 3,15). Briefly, Pol ${zeta}$ (2 nM) was incubated with 30 nM DNA substratein the presence of increasing concentrations of a single deoxynucleotidefor 10 min at 30°C. The reaction products were resolvedon 15% polyacrylamide gels containing 8 M urea. Gel band intensitiesof the substrates and products were quantitated by PhosphorImager,and the observed rate of deoxynucleotide incorporation was plottedas a function of deoxynucleotide triphosphate (dNTP) concentration.The data were fit by nonlinear regression using SigmaPlot 5.0to the Michaelis-Menten equation describing a hyperbola, v =(V_max x [dNTP]/(K_m+ [dNTP]). The k_cat and K_m steady-state parameterswere obtained from the fit and were used to calculate the frequencyof deoxynucleotide incorporation (f_inc) and the intrinsic efficiencyof mispair extension (f⁰_ext) for each primer-template pair byusing the following equation: f_inc or f⁰_ext = (k_cat/K_m)_mispaired/(k_cat/K_m)_paired.

RESULTS

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Results

Bypass of the 8-oxoG and m6G lesions by the combined action of Pol ${delta}$ and Pol ${zeta}$ . The bypass of an 8-oxoG and an m6G lesion was examined in runningstart reactions by using a 75-nt template containing an 8-oxoGor an m6G residue 45 nt from the 3' end, which was primed witha 5'-³²P-labeled 40-nt oligomer. Polymerase reactions were carriedout at low (5 µM) as well as at high (100 µM) nucleotideconcentrations to monitor lesion bypass under both restrictiveand unrestrictive conditions. First, we examined the abilityof yeast Pol ${zeta}$ to replicate past the 8-oxoG lesion in the DNAtemplate (Fig. 1A). 8-oxoG is a strong block to DNA synthesisby Pol ${zeta}$ , as even at 100 µM dNTP, compared to synthesisthrough the corresponding undamaged G residue (Fig. 1A, lane5), Pol ${zeta}$ replicated through only $~$ 12% of the 8-oxoG lesions (Fig.1A, lane 12). Although Pol ${zeta}$ was severely inhibited from insertinga nucleotide opposite 8-oxoG, which is indicated by a strongstall site right before the 8-oxoG residue, Pol ${zeta}$ was able toextend opposite from the 8-oxoG residue, which is shown by anabsence of a stall site opposite this lesion (Fig. 1A, lane12). This observation raised the possibility that Pol ${zeta}$ is ableto stimulate the bypass of 8-oxoG by extending from the nucleotidepreviously inserted opposite 8-oxoG by another DNA polymerase.Yeast Pol ${delta}$ , the replicative DNA polymerase, is able to replicatethough the 8-oxoG lesion; however, compared to replication throughan undamaged G (Fig. 1A, lanes 2 and 3), it replicates throughthe 8-oxoG lesion inefficiently (Fig. 1A, lanes 9 and 10). Remarkably,the most efficient bypass of the 8-oxoG lesion occurred whenPol ${zeta}$ was combined with Pol ${delta}$ , and these polymerases together replicatedthrough the 8-oxoG lesion almost as efficiently as through theundamaged G residue (Fig. 1A, compare lanes 13 and 14 with lanes6 and 7).

Pol ${zeta}$ replicated through an m6G residue also, but again, the bypasswas highly inefficient (Fig. 1B, compare lanes 11 and 12 withlanes 4 and 5). However, the absence of a stall site oppositem6G in contrast to a strong stall site right before this lesionindicated that Pol ${zeta}$ was able to extend the 3' primer end oppositefrom m6G, and only the nucleotide incorporation opposite m6Gwas severely inhibited (Fig. 1B, lanes 11 and 12). Pol ${delta}$ showssome ability to incorporate nucleotides opposite the m6G residue;however, it is quite inefficient in bypassing this lesion (Fig.1B, compare lanes 9 and 10 with lanes 2 and 3). Importantly,the combination of Pol ${zeta}$ with Pol ${delta}$ promoted m6G bypass, and togetherthey replicated through the m6G lesion $~$ 40% as efficiently asthrough the undamaged G residue (Fig. 1B, compare lane 14 withlane 7). The key result here is that the two DNA polymerases,Pol ${delta}$ and Pol ${zeta}$ , together are much more effective in 8-oxoG andm6G bypass than either polymerase alone. Moreover, these observationssuggested that since Pol ${delta}$ would be the first DNA polymerase toencounter the DNA lesion during its role in DNA replication,it would contribute to the insertion of nucleotides oppositethe 8-oxoG and m6G lesions, while Pol ${zeta}$ could carry out the subsequentextension step from the ensuing primer end.

Extension of primer ends situated opposite the 8-oxoG and m6G lesions by Pol ${zeta}$ . Next, we examined the ability of Pol ${zeta}$ to extend from the various3' primer termini situated across from an 8-oxoG or an m6G sitein the 75-nt template in the presence of a 100 µM concentrationof each of the four dNTPs (Fig. 2). Pol ${zeta}$ is a promiscuous extenderof primer-terminal mispairs opposite undamaged DNA templates(Fig. 2, lanes 1 to 4) and, importantly, it is also very efficientat extending from primer ends opposite from the 8-oxoG and m6Gresidues (Fig. 2, lanes 5 to 8 and lanes 9 to 12, respectively).Pol ${zeta}$ extended from an A opposite the 8-oxoG site $~$ 90% as efficientlyas from a C opposite undamaged G (Fig. 2, compare lane 6 withlane 4). The extension from a C, a G, and a T opposite from8-oxoG was also efficient, occurring with 67, 44, and 41% ofthe efficiency of extension from the C opposite undamaged G.Pol ${zeta}$ extended from a T, a G, a C, and an A opposite from them6G residue with 40 to 50% of the efficiency of extension fromthe C opposite undamaged G (Fig. 2, compare lanes 9 to 12 withlane 4).

We have previously shown that yeast Pol ${delta}$ predominantly insertsan A residue opposite 8-oxoG (6) and a T or a C opposite m6G(4). Thus, efficient bypass of 8-oxoG and m6G lesions couldbe carried out by incorporating an A residue opposite 8-oxoGand a T or a C residue opposite m6G, respectively, by Pol ${delta}$ , andby the subsequent extension of the resulting 3' primer end byPol ${zeta}$ .

Steady-state kinetic analysis of nucleotide insertion and extension reactions across from the 8-oxoG and m6G lesions by Pol ${zeta}$ . To characterize further the spectrum and the efficiencies ofnucleotide insertion and extension reactions across from 8-oxoGand m6G lesions by Pol ${zeta}$ , we measured the steady-state kineticparameters of these reactions. The kinetics of insertion ofa single deoxynucleotide opposite an 8-oxoG and an m6G and thekinetics of addition of the next correct nucleotide to various3' primer termini situated across from 8-oxoG and m6G were determinedas a function of deoxynucleotide concentration under steady-stateconditions. We incubated Pol ${zeta}$ with the 8-oxoG- and m6G-containingDNA substrates and increasing concentrations of a single nucleotide(Fig. 3). The rate of nucleotide incorporation was plotted asa function of nucleotide concentration. The steady-state apparentk_cat and K_m values for each nucleotide incorporation as wellas for the extension of each primer terminus were obtained fromthe curve fitted to the Michaelis-Menten equation by using nonlinearregression. The frequency of nucleotide incorporation, f_inc,and the relative efficiency of extension, f⁰_ext, were calculatedas the ratio of the efficiency (k_cat/K_m) of incorrect nucleotideincorporated or extended from, to the efficiency (k_cat/K_m) ofcorrect nucleotide incorporated or extended from, respectively.As shown in Table 1, Pol ${zeta}$ incorporated an A or a C opposite 8-oxoGabout 100-fold less efficiently than the incorporation of aC opposite undamaged G in the template. However, Pol ${zeta}$ extendedfrom the primer end situated opposite the 8-oxoG very efficiently(Table 2). Compared to the extension from a C opposite undamagedG in the template, Pol ${zeta}$ extended from an A, a C, a G, and a T3' primer ends situated opposite the 8-oxoG residue with frequenciesof 7.3 x 10^-1, 1.7 x 10^-1, 4.1 x 10^-2, and 4 x 10^-3, respectively(Table 2). Thus, Pol ${zeta}$ extends from an A opposite 8-oxoG aboutas efficiently as from a C opposite undamaged G in the template.

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TABLE 1. Steady-state kinetic parameters of nucleotide insertion reactions opposite 8-oxoG and m6G template residues by yeast Pol ${zeta}$

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TABLE 2. Steady-state kinetic parameters of extension reactions opposite from 8-oxoG and m6G template residues by yeast Pol ${zeta}$

Pol ${zeta}$ is also very inefficient at incorporating nucleotides oppositethe m6G lesion. Pol ${zeta}$ incorporated nucleotides opposite m6G withfrequencies ranging from 1.4 x 10^-3 to 4.4 x 10^-5, and eventhe incorporation of a T, the most efficiently incorporatednucleotide opposite m6G by Pol ${zeta}$ , was $~$ 700-fold less efficientthan the incorporation of C opposite undamaged G (Table 1).In contrast, Pol ${zeta}$ extended the primer ends opposite from them6G lesion quite efficiently. Pol ${zeta}$ extended from a C oppositem6G $~$ 5-fold less efficiently than from a C opposite an undamagedG in the template and, intriguingly, Pol ${zeta}$ could also extend froma G placed opposite m6G quite efficiently. Overall, the steady-statekinetic analyses indicate that Pol ${zeta}$ is quite inefficient in insertingnucleotides opposite the 8-oxoG and m6G lesions, but it is quiteefficient at extending primer ends opposite from these lesions.

DISCUSSION

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Discussion

We have shown previously that Pol ${zeta}$ is highly inefficient at insertingnucleotides opposite the 3' T of a cis-syn TT dimer or a (6-4)TT photoproduct and also that it is unable to insert nucleotidesopposite an abasic site (5, 7, 11). The inefficient abilityof Pol ${zeta}$ to incorporate nucleotides opposite a CPD or (6-4) photoproductcould be explained by its inability to accommodate these distortinglesions in its active site, and the inability to incorporatenucleotides opposite an abasic site may suggest the need ofPol ${zeta}$ for a templating base. Here, we show that Pol ${zeta}$ is also veryinefficient at inserting nucleotides opposite the 8-oxoG andm6G lesions. This is rather surprising, given the fact thatPol ${delta}$ can insert an A opposite 8-oxoG and a T or a C oppositem6G (4, 6). Compared to the insertion of a C opposite undamagedtemplate G, Pol ${zeta}$ shows an $~$ 100-fold-reduced efficiency for theinsertion of an A opposite 8-oxoG, and it inserts a T or a Copposite the m6G lesion with an efficiency that is reduced by $~$ 700-fold or more. The highly inefficient incorporation of nucleotidesopposite the 8-oxoG or m6G lesions by Pol ${zeta}$ , where Pol ${delta}$ is ableto insert the nucleotides which cause the least amount of geometricdistortion, suggests that Pol ${zeta}$ is very sensitive to any modificationof the templating base.

By contrast to its high degree of sensitivity to geometric distortionsof the templating base, Pol ${zeta}$ can tolerate a wide range of distortionsat the primer terminus. Thus, it can efficiently extend frommispaired primer termini on undamaged as well as on damagedDNAs. Here we show that Pol ${zeta}$ proficiently extends from an A opposite8-oxoG, which is the nucleotide inserted opposite this lesionby Pol ${delta}$ . Accordingly, the most proficient replication throughthe 8-oxoG lesion occurs when Pol ${delta}$ is combined with Pol ${zeta}$ . Thus,Pol ${zeta}$ could contribute to the mutagenic bypass of this DNA lesion.Pol ${zeta}$ extends from a C opposite m6G only $~$ 5-fold less well thanit extends from a C opposite undamaged G. Pol ${zeta}$ is, however, lessefficient ( $~$ 30-fold) at extending from a T opposite m6G, butPol ${zeta}$ extends quite well from a G opposite this lesion. Thus,Pol ${zeta}$ could contribute to the error-free bypass of m6G lesionsby extending from the C nucleotide inserted by Pol ${delta}$ . For them6G lesion also, the most proficient bypass occurs in the presenceof both Pol ${delta}$ and Pol ${zeta}$ .

Yeast Pol ${delta}$ exhibits a strong stall site just before the 8-oxoGand m6G lesions, suggesting that this polymerase is inhibitedat the nucleotide incorporation step. It is possible that someof this block derives from the subsequent removal of the incorporatednucleotide by Pol ${delta}$ 's 3' $->$ 5' exonuclease activity. In that case,in the presence of Pol ${zeta}$ , because of the proficient extensionof the primer terminus opposite the lesion, the forward synthesisreaction could counteract the backward exonucleolytic reactionof Pol ${delta}$ , and that may account for the synergistic enhancementof lesion bypass that occurs when Pol ${delta}$ is combined with Pol ${zeta}$ .

The results with the DNA lesions studied so far indicate thatthe role of Pol ${zeta}$ is to extend from the nucleotide inserted oppositethe lesion site by another DNA polymerase from which the inserterpolymerase is unable to extend (Table 3). Thus, for a TT dimer,Pol ${eta}$ mediates the error-free bypass of this lesion by insertingan A opposite the 3' T and then extending the primer end byinserting an A opposite the 5' T of the lesion. However, ifa wrong nucleotide, such as a G, were to be inserted oppositethe 3' T by Pol ${eta}$ , then the extension step would become dependentupon Pol ${zeta}$ because of its more proficient ability to extend fromthis primer terminus (11) than that of Pol ${eta}$ (19). Opposite the3' T of a (6-4) TT photoproduct, Pol ${eta}$ inserts a G about eightfoldmore efficiently than an A, but it cannot extend from eitherof these nucleotides (7). Pol ${zeta}$ is $~$ 3-fold more efficient at extendingfrom a G than from an A opposite this lesion (7, 11). Thus,the sequential action of Pol ${eta}$ and Pol ${zeta}$ could effect the mutagenicbypass of a (6-4) TT photoproduct (Table 3). In fact, geneticstudies in yeast have corroborated the involvement of Pol ${eta}$ inthe mutagenic bypass of the (6-4) TT lesion, as the incidenceof 3' T $->$ C mutations in a plasmid carrying a site-specific (6-4)TT lesion is greatly reduced in the absence of Pol ${eta}$ (1). Becauseof the inability of any other polymerase to extend from thenucleotide inserted opposite the 3' T of a (6-4) photoproduct,TLS through this lesion, whether mutagenic or error free, wouldbe entirely dependent upon Pol ${zeta}$ (Table 3). Similarly, TLS throughan abasic site would also have the absolute requirement of Pol ${zeta}$ for the extension step, as no other polymerase is able to performthis task (Table 3).

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TABLE 3. Role of yeast Pol ${zeta}$ in extending primer ends opposite from DNA lesions

Although yeast Pol ${delta}$ can replicate through the 8-oxoG lesion,it stalls at both the insertion and extension steps (6) (Fig.1A); therefore, we expect it to make only a marginal contributionto the bypass of this DNA lesion. In yeast, Pol ${eta}$ plays a predominantrole in the error-free bypass of 8-oxoG (Table 3), as the frequencyof G:C to T:A transversions is greatly increased in a strainlacking both Pol ${eta}$ and the Ogg1 DNA glycosylase which functionsin the removal of 8-oxoG paired with C (6). The increased frequencyof G:C to T:A transversions that ensue from the replicativebypass of 8-oxoG presumably result from the insertion of anA opposite this lesion by Pol ${delta}$ (6). Although either Pol ${eta}$ or Pol ${zeta}$ could extend from the A nucleotide inserted opposite 8-oxoGby Pol ${delta}$ (Table 3), we expect Pol ${zeta}$ to be more effective at thisstep since it is almost as efficient at extending from the Aopposite 8-oxoG as it is in extending from a C opposite G, whereasPol ${eta}$ extends from an A opposite 8-oxoG with an $~$ 6-fold-reducedefficiency (6).

Genetic studies in yeast have indicated the requirement of Pol ${eta}$ and Pol ${delta}$ for the mutagenic bypass of the m6G lesion (4). WhilePol ${eta}$ replicates through the m6G lesion fairly efficiently byinserting a C or T residue and by extending from the insertednucleotide, Pol ${delta}$ is quite inefficient at bypassing this lesion,as it is inhibited at both the insertion and extension steps(4) (Fig. 1B). However, because of the proximity of Pol ${delta}$ to thelesion site, it could still be able to carry out the nucleotideincorporation step but stall at the extension step. In thatcase, the subsequent extension could depend upon Pol ${zeta}$ or Pol ${eta}$ (Table 3).

In summary, Pol ${zeta}$ is very adept at extending primer ends oppositefrom a diverse array of DNA lesions. It is the only yeast polymerasethat is able to proficiently extend from nucleotides insertedopposite a (6-4) dipyrimidine photoproduct or an abasic site,which explains its in vivo requirement for TLS through theseDNA lesions (10, 16). For the extension of primer ends oppositefrom the 8-oxoG and m6G lesions, however, Pol ${zeta}$ could competewith Pol ${eta}$ . Pol ${zeta}$ and Pol ${eta}$ differ remarkably in their roles in lesionbypass, as Pol ${zeta}$ is a highly specialized polymerase that actsprimarily to extend the primer ends opposite from DNA lesions,regardless of the degree of geometric distortion they cause,whereas Pol ${eta}$ bypasses moderately distorting lesions by carryingout both the insertion and extension steps.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Health grantGM19261.

FOOTNOTES

* Corresponding author. Mailing address: Sealy Center for Molecular Science, University of Texas Medical Branch, 6.104 Blocker Medical Research Building, 11th and Mechanic Streets, Galveston, TX 77555-1061. Phone: (409) 747-8601. Fax: (409) 747-8608. E-mail: l.prakash{at}utmb.edu.

REFERENCES

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