Mutator alleles of yeast DNA polymerase zeta

The yeast REV3 gene encodes the catalytic subunit of DNA polymerase zeta (pol zeta), a B family polymerase that performs mutagenic DNA synthesis in cells. To probe pol zeta mutagenic functions, we generated six mutator alleles of REV3 with amino acid replacements for Leu979, a highly conserved residue inferred to be at the pol zeta active site. Replacing Leu979 with Gly, Val, Asn, Lys, Met or Phe resulted in yeast strains with elevated UV-induced mutant frequencies. While four of these strains had reduced survival following UV irradiation, the rev3-L979F and rev3-L979M strains had normal survival, suggesting retention of pol zeta catalytic activity. UV mutagenesis in the rev3-L979F background was increased when photoproduct bypass by pol eta was eliminated by deletion of RAD30. The rev3-L979F mutation had little to no effect on mutagenesis in an ogg1Delta background, which cannot repair 8-oxo-guanine in DNA. UV-induced can1 mutants from rev3-L979F and rad30Deltarev3-L979F strains primarily contained base substitutions and complex mutations, suggesting error-prone bypass of UV photoproducts by L979F pol zeta. Spontaneous mutation rates in rev3-L979F and rev3-L979M strains are elevated by about two-fold overall and by two- to eight-fold for C to G transversions and complex mutations, both of which are known to be generated by wild-type pol zetain vitro. These results indicate that Rev3p-Leu979 replacements reduce the fidelity of DNA synthesis by yeast pol zetain vivo. In conjunction with earlier studies, the data establish that the conserved amino acid at the active site location occupied by Leu979 is critical for the fidelity of all four yeast B family polymerases. Reduced fidelity with retention of robust polymerase activity suggests that the homologous rev3-L979F allele may be useful for analyzing pol zeta functions in mammals, where REV3 deletion is lethal.     

Frameshifts and deletions during in vitro translesion synthesis past Pt-DNA adducts by DNA polymerases beta and eta

DNA polymerases beta (pol beta ) and eta (pol eta ) are the only two eukaryotic polymerases known to efficiently bypass cisplatin and oxaliplatin adducts in vitro. Frameshift errors are an important aspect of mutagenesis. We have compared the types of frameshifts that occur during translesion synthesis past cisplatin and oxaliplatin adducts in vitro by pol beta and pol eta on a template containing multiple runs of nucleotides flanking a single platinum-GG adduct. Translesion synthesis past platinum adducts by pol beta resulted in approximately 50% replication products containing single-base deletions. For both adducts the majority of -1 frameshifts occurred in a TTT sequence 3-5 bp upstream of the DNA lesion. For pol eta, all of the bypass products for both cisplatin and oxaliplatin adducts contained -1 frameshifts in the upstream TTT sequence and most of the products of replication on oxaliplatin-damaged templates had multiple replication errors, both frameshifts and misinsertions. In addition, on platinated templates both polymerases generated replication products 4-8 bp shorter than the full-length products. The majority of short cisplatin-induced products contained an internal deletion which included the adduct. In contrast, the majority of oxaliplatin-induced short products contained a 3' terminal deletion. The implications of these in vitro results for in vivo mutagenesis are discussed.     

Efficient translesion replication past oxaliplatin and cisplatin GpG adducts by human DNA polymerase eta

Platinum anticancer agents form bulky DNA adducts which are thought to exert their cytotoxic effect by blocking DNA replication. Translesion synthesis, one of the pathways of postreplication repair, is thought to account for some resistance to DNA damage and much of the mutagenicity of bulky DNA adducts in dividing cells. Oxaliplatin has been shown to be effective in cisplatin-resistant cell lines and less mutagenic than cisplatin in the Ames assay. We have shown that the eukaryotic DNA polymerases yeast pol zeta, human pol beta, and human pol gamma bypass oxaliplatin-GG adducts more efficiently than cisplatin-GG adducts. Human pol eta, a product of the XPV gene, has been shown to catalyze efficient translesion synthesis past cis, syn-cyclobutane pyrimidine dimers. In the present study we compared translesion synthesis past different Pt-GG adducts by human pol eta. Our data show that, similar to other eukaryotic DNA polymerases, pol eta bypasses oxaliplatin-GG adducts more efficiently than cisplatin-GG adducts. However, pol eta-catalyzed translesion replication past Pt-DNA adducts was more efficient and less accurate than that seen for previously tested polymerases. We show that the efficiency and fidelity of translesion replication past Pt-DNA adducts appear to be determined by both the structure of the adduct and the DNA polymerase active site.     

Proofreading of DNA polymerase eta-dependent replication errors

Human DNA polymerase eta, the product of the skin cancer susceptibility gene XPV, bypasses UV photoproducts in template DNA that block synthesis by other DNA polymerases. Pol eta lacks an intrinsic proofreading exonuclease and copies DNA with low fidelity, such that pol eta errors could contribute to mutagenesis unless they are corrected. Here we provide evidence that pol eta can compete with other human polymerases during replication of duplex DNA, and in so doing it lowers replication fidelity. However, we show that pol eta has low processivity and extends mismatched primer termini less efficiently than matched termini. These properties could provide an opportunity for extrinsic exonuclease(s) to proofread pol eta-induced replication errors. When we tested this hypothesis during replication in human cell extracts, pol eta-induced replication infidelity was found to be modulated by changing the dNTP concentration and to be enhanced by adding dGMP to a replication reaction. Both effects are classical hallmarks of exonucleolytic proofreading. Thus, pol eta is ideally suited for its role in reducing UV-induced mutagenesis and skin cancer risk, in that its relaxed base selectivity may facilitate efficient bypass of UV photoproducts, while subsequent proofreading by extrinsic exonuclease(s) may reduce its mutagenic potential.     

Effects of accessory proteins on the bypass of a cis-syn thymine-thymine dimer by Saccharomyces cerevisiae DNA polymerase eta

Among several hypotheses to explain how translesion synthesis (TLS) by DNA polymerase eta (pol eta) suppresses ultraviolet light-induced mutagenesis in vivo despite the fact that pol eta copies DNA with low fidelity, here we test whether replication accessory proteins enhance the fidelity of TLS by pol eta. We first show that the single-stranded DNA binding protein RPA, the sliding clamp PCNA, and the clamp loader RFC slightly increase the processivity of yeast pol eta and its ability to recycle to new template primers. However, these increases are small, and they are similar when copying an undamaged template and a template containing a cis-syn TT dimer. Consequently, the accessory proteins do not strongly stimulate the already robust TT dimer bypass efficiency of pol eta. We then perform a comprehensive analysis of yeast pol eta fidelity. We show that it is much less accurate than other yeast DNA polymerases and that the accessory proteins have little effect on fidelity when copying undamaged templates or when bypassing a TT dimer. Thus, although accessory proteins clearly participate in pol eta functions in vivo, they do not appear to help suppress UV mutagenesis by improving pol eta bypass fidelity per se.     

Efficiency of extension of mismatched primer termini across from cisplatin and oxaliplatin adducts by human DNA polymerases beta and eta in vitro

DNA polymerases beta and eta are among the few eukaryotic polymerases known to efficiently bypass cisplatin and oxaliplatin adducts in vitro. Our laboratory has previously established that both polymerases misincorporated dTTP with high frequency across from cisplatin- and oxaliplatin-GG adducts. This decrease in polymerase fidelity on platinum-damaged DNA could lead to in vivo mutations, if this base substitution were efficiently elongated. In this study, we performed a steady-state kinetic analysis of the steps required for fixation of dTTP misinsertion during translesion synthesis past cisplatin- and oxaliplatin-GG adducts by pol beta and pol eta. The efficiency of translesion synthesis by pol eta past Pt-GG adducts was very similar to that observed for this polymerase when the template contains thymine-thymine dimers. This finding suggested that pol eta could play a role in translesion synthesis past platinum-GG adducts in vivo. On the other hand, translesion synthesis past platinum-GG adducts by pol beta was much less efficient. Translesion synthesis by pol eta is likely to be predominantly error-free, since the probability of correct insertion and extension by pol eta was 1000-2000-fold greater than the probability of incorrect insertion and extension. Our results also indicated that for pol eta the frequency of misincorporation is the same across from the 3'G and the 5'G of the platinum-GG adducts for both cisplatin and oxaliplatin adducts. On the other hand, pol beta is more likely to misinsert at the 3'G of the adducts and misinsertion occurs at higher frequency for oxaliplatin-GG than for cisplatin-GG adducts.     

The in vivo characterization of translesion synthesis across UV-induced lesions in Saccharomyces cerevisiae: insights into Pol zeta- and Pol eta-dependent frameshift mutagenesis

UV irradiation, a known carcinogen, induces the formation of dipyrimidine dimers with the predominant lesions being cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone adducts (6-4PPs). The relative roles of the yeast translesion synthesis DNA polymerases Pol zeta and Pol eta in UV survival and mutagenesis were examined using strains deficient in one or both polymerases. In addition, photoreactivation was used to specifically remove CPDs, thus allowing an estimate to be made of the relative contributions of CPDs vs. 6-4PPs to overall survival and mutagenesis. In terms of UV-induced mutagenesis, we focused on the +1 frameshift mutations detected by reversion of the lys2deltaA746 allele, as Pol zeta produces a distinct mutational signature in this assay. Results suggest that CPDs are responsible for most of the UV-associated toxicity as well as for the majority of UV-induced frameshift mutations in yeast. Although the presence of Pol eta generally suppresses UV-induced mutagenesis, our data suggest a role for this polymerase in generating some classes of +1 frameshifts. Finally, the examination of frameshift reversion spectra indicates a hierarchy between Pol eta and Pol zeta with respect to the bypass of UV-induced lesions.     

The relative roles in vivo of Saccharomyces cerevisiae Pol eta, Pol zeta, Rev1 protein and Pol32 in the bypass and mutation induction of an abasic site, T-T (6-4) photoadduct and T-T cis-syn cyclobutane dimer

We have investigated the relative roles in vivo of Saccharomyces cerevisiae DNA polymerase eta, DNA polymerase zeta, Rev1 protein, and the DNA polymerase delta subunit, Pol32, in the bypass of an abasic site, T-T (6-4) photoadduct and T-T cis-syn cyclobutane dimer, by transforming strains deleted for RAD30, REV3, REV1, or POL32 with duplex plasmids carrying one of these DNA lesions located within a 28-nucleotide single-stranded region. DNA polymerase eta was found to be involved only rarely in the bypass of the T-T (6-4) photoadduct or the abasic sites in the sequence context used, although, as expected, it was solely responsible for the bypass of the T-T dimer. We argue that DNA polymerase zeta, rather than DNA polymerase delta as previously suggested, is responsible for insertion in bypass events other than those in which polymerase eta performs this function. However, DNA polymerase delta is involved indirectly in mutagenesis, since the strain lacking its Pol32 subunit, known to be deficient in mutagenesis, shows as little bypass of the T-T (6-4) photoadduct or the abasic sites as those deficient in Pol zeta or Rev1. In contrast, bypass of the T-T dimer in the pol32delta strain occurs at the wild-type frequency.     

Rev1 enhances CAG.CTG repeat stability in Saccharomyces cerevisiae

Trinucleotide repeats (TNRs) frequently expand in certain human genetic diseases, often with devastating pathological consequences. TNR expansions require the addition of new DNA; accordingly, molecular models suggest aberrant DNA replication or error-prone repair synthesis as the sources of most instability. Some proteins are currently known that either promote or inhibit TNR mutability. To identify additional proteins that help protect cells against TNR instability, yeast mutants were isolated with higher than normal rates of CAG.CTG tract expansions. Surprisingly, a rev1 mutant was isolated. In contrast to its canonical function in supporting mutagenesis, we found that Rev1 reduces rates of CAG.CTG repeat expansions and contractions, as judged by the behavior of the rev1 mutant. The rev1 mutator phenotype was specific for TNRs with hairpin forming capacity. Mutations in REV3 or REV7, encoding the subunits of DNA polymerase zeta (pol zeta), did not affect expansion rates in REV1 or rev1 strains. A rev1 point mutant lacking dCMP transferase activity was normal for TNR instability, whereas the rev1-1 allele that interferes with BRCT domain function was as defective as a rev1 null mutant. In summary, these results indicate that yeast Rev1 reduces mutability of CAG.CTG tracts in a manner dependent on BRCT domain function but independent of dCMP transferase activity and of pol zeta.     

Mammalian translesion DNA synthesis across an acrolein-derived deoxyguanosine adduct. Participation of DNA polymerase eta in error-prone synthesis in human cells

alpha-OH-PdG, an acrolein-derived deoxyguanosine adduct, inhibits DNA synthesis and miscodes significantly in human cells. To probe the cellular mechanism underlying the error-free and error-prone translesion DNA syntheses, in vitro primer extension experiments using purified DNA polymerases and site-specific alpha-OH-PdG were conducted. The results suggest the involvement of pol eta in the cellular error-prone translesion synthesis. Experiments with xeroderma pigmentosum variant cells, which lack pol eta, confirmed this hypothesis. The in vitro results also suggested the involvement of pol iota and/or REV1 in inserting correct dCMP opposite alpha-OH-PdG during error-free synthesis. However, none of translesion-specialized DNA polymerases catalyzed significant extension from a dC terminus when paired opposite alpha-OH-PdG. Thus, our results indicate the following. (i) Multiple DNA polymerases are involved in the bypass of alpha-OH-PdG in human cells. (ii) The accurate and inaccurate syntheses are catalyzed by different polymerases. (iii) A modification of the current eukaryotic bypass model is necessary to account for the accurate bypass synthesis in human cells.     

Cutting edge: DNA polymerases mu and lambda are dispensable for Ig gene hypermutation

Mutations arising in Ig V genes during an immune response are most likely introduced by one or several error-prone DNA polymerases. Many of the recently described nonreplicative DNA polymerases have an intrinsic fidelity compatible with such an activity, the strongest candidates being polymerase (pol) eta, pol iota, pol zeta, and pol mu. We report in this work that mice inactivated for either of the two polymerases related to pol beta (i.e., pol mu and pol lambda) are viable and fertile and display a normal hypermutation pattern.     

Proliferating cell nuclear antigen promotes translesion synthesis by DNA polymerase zeta

DNA polymerase zeta (Pol zeta), a heterodimer of Rev3 and Rev7, is essential for DNA damage provoked mutagenesis in eukaryotes. DNA polymerases that function in a processive complex with the replication clamp proliferating cell nuclear antigen (PCNA) have been shown to possess a close match to the consensus PCNA-binding motif QxxLxxFF. This consensus motif is lacking in either subunit of Pol zeta, yet its activity is stimulated by PCNA. In particular, translesion synthesis of UV damage-containing DNA is dramatically stimulated by PCNA such that translesion synthesis rates are comparable with replication rates by Pol zeta on undamaged DNA. PCNA also stimulated translesion synthesis of a model abasic site by Pol zeta. Efficient PCNA stimulation required that PCNA was prevented from sliding off the damage-containing model oligonucleotide template-primer through the use of biotin-streptavidin bumpers or other blocks. Under those experimental conditions, facile bypass of the abasic site was also detected by DNA polymerase delta or eta (Rad30). The yeast DNA damage checkpoint clamp, consisting of Rad17, Mec3, and Ddc1, and an ortholog of human 9-1-1, has been implicated in damage-induced mutagenesis. However, this checkpoint clamp did not stimulate translesion synthesis by Pol zeta or by DNA polymerase delta.     

Mutagenic and recombinagenic responses to defective DNA polymerase delta are facilitated by the Rev1 protein in pol3-t mutants of Saccharomyces cerevisiae

Defective DNA replication can result in substantial increases in the level of genome instability. In the yeast Saccharomyces cerevisiae, the pol3-t allele confers a defect in the catalytic subunit of replicative DNA polymerase delta that results in increased rates of mutagenesis, recombination, and chromosome loss, perhaps by increasing the rate of replicative polymerase failure. The translesion polymerases Pol eta, Pol zeta, and Rev1 are part of a suite of factors in yeast that can act at sites of replicative polymerase failure. While mutants defective in the translesion polymerases alone displayed few defects, loss of Rev1 was found to suppress the increased rates of spontaneous mutation, recombination, and chromosome loss observed in pol3-t mutants. These results suggest that Rev1 may be involved in facilitating mutagenic and recombinagenic responses to the failure of Pol delta. Genome stability, therefore, may reflect a dynamic relationship between primary and auxiliary DNA polymerases.     

trans-Lesion synthesis past bulky benzo[a]pyrene diol epoxide N2-dG and N6-dA lesions catalyzed by DNA bypass polymerases

The effectiveness of in vitro primer elongation reactions catalyzed by human bypass DNA polymerases kappa (hDinB1), pol eta (hRad30A), pol iota (hRad30B), and yeast pol zeta (Rev3 and Rev7) in site-specifically modified template oligonucleotide strands were studied in vitro. The templates contained single bulky lesions derived from the trans-addition of the mutagenic (+)- or (-)-enantiomers of r7,t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (a metabolite of the environmental carcinogen benzo[a]pyrene), to the exocyclic amino groups of guanine or adenine in oligonucleotide templates 33, or more, bases long. In "running start" primer extension reactions, pol kappa effectively bypassed both the stereoisomeric (+)- and (-)-trans-guanine adducts but not the analogous adenine adducts. In sharp contrast, pol eta, which exhibits considerable sequence homology with pol kappa (both belong to the group of Y family polymerases), is partially blocked by the guanine adducts and the (-)-trans-adenine adduct, although the stereoisomeric (+)-trans-adenine adduct is more successfully bypassed. Neither pol iota nor pol zeta, either alone or in combination, were effective in trans-lesion synthesis past the same adducts. In all cases, the fidelity of insertion is dependent on adduct stereochemistry and structure. Generally, error-free nucleotide insertion opposite the lesions tends to depend more on adduct stereochemistry than error-prone insertion. None of the polymerases tested are a universal bypass polymerase for the stereoisomeric bulky polycyclic aromatic hydrocarbon-DNA adducts derived from anti-BPDE.     

Amino acid substitutions at conserved tyrosine 52 alter fidelity and bypass efficiency of human DNA polymerase eta

DNA polymerase eta (Pol eta) is a member of a new class of DNA polymerases that is able to copy DNA containing damaged nucleotides. These polymerases are highly error-prone during copying of unaltered DNA templates. We analyzed the relationship between bypass efficiency and fidelity of DNA synthesis by introducing substitutions for Tyr-52, a highly conserved amino acid, within the human DNA polymerase eta (hPol eta) finger domain. Most substitutions for Tyr-52 caused reduction in bypass of UV-associated damage, measured by the ability to rescue the viability of UV-sensitive yeast cells at a high UV dose. For most mutants, the reduction in bypass ability paralleled the reduction in polymerization activity. Interestingly, the hPol eta Y52E mutant exhibited a greater reduction in bypass efficiency than polymerization activity. The reduction in bypass efficiency was accompanied by an up to 11-fold increase in the incorporation of complementary nucleotides relative to non-complementary nucleotides. The fidelity of DNA synthesis, measured by copying a gapped M13 DNA template in vitro, was also enhanced as much as 15-fold; the enhancement resulted from a decrease in transitions, which were relatively frequent, and a large decrease in transversions. Our demonstration that an amino acid substitution within the active site enhances the fidelity of DNA synthesis by hPol eta, one of the most inaccurate of DNA polymerases, supports the hypothesis that even error-prone DNA polymerases function in base selection.     

Substitution of a residue contacting the triphosphate moiety of the incoming nucleotide increases the fidelity of yeast DNA polymerase zeta

DNA polymerase zeta (pol zeta), which is required for DNA damage-induced mutagenesis, functions in the error-prone replication of a wide range of DNA lesions. During this process, pol zeta extends from nucleotides incorporated opposite template lesions by other polymerases. Unlike classical polymerases, pol zeta efficiently extends from primer-terminal base pairs containing mismatches or lesions, and it synthesizes DNA with moderate fidelity. Here we describe genetic and biochemical studies of three yeast pol zeta mutant proteins containing substitutions of highly conserved amino acid residues that contact the triphosphate moiety of the incoming nucleotide. The R1057A and K1086A proteins do not complement the rev3Delta mutation, and these proteins have significantly reduced polymerase activity relative to the wild-type protein. In contrast, the K1061A protein partially complements the rev3Delta mutation and has nearly normal polymerase activity. Interestingly, the K1061A protein has increased fidelity relative to wild-type pol zeta and is somewhat less efficient at extending from mismatched primer-terminal base pairs. These findings have important implications both for the evolutionary divergence of pol zeta from classical polymerases and for the mechanism by which this enzyme accommodates distortions in the DNA caused by mismatches and lesions.     

Role of DNA polymerase eta in the UV mutation spectrum in human cells

In humans, inactivation of the DNA polymerase eta gene (pol eta) results in sunlight sensitivity and causes the cancer-prone xeroderma pigmentosum variant syndrome (XP-V). Cells from XP-V individuals have a reduced capacity to replicate UV-damaged DNA and show hypermutability after UV exposure. Biochemical assays have demonstrated the ability of pol eta to bypass cis-syn-cyclobutane thymine dimers, the most common lesion generated in DNA by UV. In most cases, this bypass is error-free. To determine the actual requirement of pol eta in vivo, XP-V cells (XP30RO) were complemented by the wild type pol eta gene. We have used two pol eta-corrected clones to study the in vivo characteristics of mutations produced by DNA polymerases during DNA synthesis of UV-irradiated shuttle vectors transfected into human host cells, which had or had not been exposed previously to UV radiation. The functional complementation of XP-V cells by pol eta reduced the mutation frequencies both at CG and TA base pairs and restored UV mutagenesis to a normal level. UV irradiation of host cells prior to transfection strongly increased the mutation frequency in undamaged vectors and, in addition, especially in the pol eta-deficient XP30RO cells at 5'-TT sites in UV-irradiated plasmids. These results clearly show the protective role of pol eta against UV-induced lesions and the activation by UV of pol eta-independent mutagenic processes.     

The efficiency and specificity of apurinic/apyrimidinic site bypass by human DNA polymerase eta and Sulfolobus solfataricus Dpo4

One of the most common DNA lesions arising in cells is an apurinic/apyrimidinic (AP) site resulting from base loss. Although a template strand AP site impedes DNA synthesis, translesion synthesis (TLS) DNA polymerases can bypass an AP site. Because this bypass is expected to be highly mutagenic because of loss of base coding potential, here we quantify the efficiency and the specificity of AP site bypass by two Y family TLS enzymes, Sulfolobus solfataricus DNA polymerase 4 (Dpo4) and human DNA polymerase eta (Pol eta). During a single cycle of processive DNA synthesis, Dpo4 and Pol eta bypass synthetic AP sites with 13-30 and 10-13%, respectively, of the bypass efficiency for undamaged bases in the same sequence contexts. These efficiencies are higher than for the A family, exonuclease-deficient Klenow fragment of Escherichia coli DNA polymerase I. We then determined AP site bypass specificity for complete bypass, requiring insertion or misalignment at the AP site followed by multiple incorporations using the aberrant primer templates. Although Dpo4, Pol eta, and Klenow polymerase have different fidelity when copying undamaged DNA, bypass of AP sites lacking A or G by all three polymerases is nearly 100% mutagenic. The majority (70-80%) of bypass events made by all three polymerases are insertion of dAMP opposite the AP site. Single base deletion errors comprise 10-25% of bypass events, with other base insertions observed at lower rates. Given that mammalian cells contain five polymerases implicated in TLS, and given that a large number of AP sites are generated per mammalian cell per day, even moderately efficient AP site bypass could be a source of substitution and frameshift mutagenesis in vivo.     

Translesion DNA synthesis catalyzed by human pol eta and pol kappa across 1,N6-ethenodeoxyadenosine

1,N(6)-Ethenodeoxyadenosine, a DNA adduct generated by exogenous and endogenous sources, severely blocks DNA synthesis and induces miscoding events in human cells. To probe the mechanism for in vivo translesion DNA synthesis across this adduct, in vitro primer extension studies were conducted using newly identified human DNA polymerases (pol) eta and kappa, which have been shown to catalyze translesion DNA synthesis past several DNA lesions. Steady-state kinetic analyses and analysis of translesion products have revealed that the synthesis is >100-fold more efficient with pol eta than with pol kappa and that both error-free and error-prone syntheses are observed with these enzymes. The miscoding events include both base substitution and frameshift mutations. These results suggest that both polymerases, particularly pol eta, may contribute to the translesion DNA synthesis events observed for 1,N(6)-ethenodeoxyadenosine in human cells.