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.     

NGS-based analysis of base-substitution signatures created by yeast DNA polymerase eta and zeta on undamaged and abasic DNA templates in vitro

Translesion synthesis (TLS) is the mechanism in which DNA polymerases (TLS polymerases) bypass unrepaired template damage with high error rates. DNA polymerase η and ζ (Polη and Polζ) are major TLS polymerases that are conserved from yeast to humans. In this study, we quantified frequencies of base-substitutions by yeast Polη and Polζ on undamaged and abasic templates in vitro. For accurate quantification, we used a next generation sequencing (NGS)-based method where DNA products were directly analyzed by parallel sequencing. On undamaged templates, Polη and Polζ showed distinct base-substitution profiles, and the substitution frequencies were differently influenced by the template sequence. The base-substitution frequencies were influenced mainly by the adjacent bases both upstream and downstream of the substitution sites. Thus we present the base-substitution signatures of these polymerases in a three-base format. On templates containing abasic sites, Polη created deletions at the lesion in more than 50% of the TLS products, but the formation of the deletions was suppressed by the presence of Polζ. Polζ and Polη cooperatively facilitated the TLS reaction over an abasic site in vitro, suggesting that these two polymerases can cooperate in efficient and high fidelity TLS.     

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.     

Quantitative analysis of translesion DNA synthesis across a benzo[a]pyrene-guanine adduct in mammalian cells: the role of DNA polymerase kappa

Replication across unrepaired DNA lesions in mammalian cells is effected primarily by specialized, low fidelity DNA polymerases. We studied translesion DNA synthesis (TLS) across a benzo[a]pyrene-guanine (BP-G) adduct, a major mutagenic DNA lesion generated by tobacco smoke. This was done using a quantitative assay that measures TLS indirectly, by measuring the recovery of gapped plasmids transfected into cultured mammalian cells. Analysis of PolK(+/+) mouse embryo fibroblasts (MEFs) showed that TLS across the BP-G adduct occurred with an efficiency of 48 +/- 4%, which is an order of magnitude higher than in Escherichia coli. In PolK(-/-) MEFs, bypass was 16 +/- 1%, suggesting that at least two-thirds of the BP-G adducts in MEFs were bypassed exclusively by polymerase kappa (polkappa). In contrast, poleta was not required for bypass across BP-G in a human XP-V cell line. Analysis of misinsertion specificity across BP-G revealed that bypass was more error-prone in MEFs lacking polkappa. Expression of polkappa from a plasmid introduced into PolK(-/-) MEFs restored both the extent and fidelity of bypass across BP-G. Polkappa was not required for bypass of a synthetic abasic site. In vitro analysis demonstrated efficient bypass across BP-G by both polkappa and poleta, suggesting that the biological role of polkappa in TLS across BP-G is due to regulation of TLS and not due to an exclusive ability to bypass this lesion. These results indicate that BP-G is bypassed in mammalian cells with relatively high efficiency and that polkappa bypasses BP-G in vivo with higher efficiency and higher accuracy than other DNA polymerases.     

Two‐polymerase mechanisms dictate error‐free and error‐prone translesion DNA synthesis in mammals

DNA replication across blocking lesions occurs by translesion DNA synthesis (TLS), involving a multitude of mutagenic DNA polymerases that operate to protect the mammalian genome. Using a quantitative TLS assay, we identified three main classes of TLS in human cells: two rapid and error‐free, and the third slow and error‐prone. A single gene, REV3L, encoding the catalytic subunit of DNA polymerase ζ (polζ), was found to have a pivotal role in TLS, being involved in TLS across all lesions examined, except for a TT cyclobutane dimer. Genetic epistasis siRNA analysis indicated that discrete two‐polymerase combinations with polζ dictate error‐prone or error‐free TLS across the same lesion. These results highlight the central role of polζ in both error‐prone and error‐free TLS in mammalian cells, and show that bypass of a single lesion may involve at least three different DNA polymerases, operating in different two‐polymerase combinations.     

Identification of a strand-related bias in the PCNA-mediated bypass of spontaneous lesions by yeast Poleta

Translesion synthesis (TLS) DNA polymerases are specialized to bypass lesions that block replicative polymerases and prevent complete genome duplication. Current TLS models hypothesize that PCNA, the polymerase processivity clamp, is important for regulating the access and loading of the low fidelity TLS polymerases onto DNA in response to replication-blocking lesions. PCNA binds to the C-terminus of yeast Poleta, for example, and this interaction is required for cell survival after UV irradiation. Previously, we identified two spontaneous, Polzeta-dependent "complex" mutation hotspots using the lys2DeltaA746 frameshift reversion assay in repair-compromised cells. In the current study we observed an accumulation of Polzeta-dependent complex frameshifts at a third hotspot in Poleta-deficient cells. Interestingly, the sequence of this third hotspot is the reverse complement of the two hotspots previously identified, suggesting that the utilization of Polzeta and Poleta may be related to the position of the relevant lesion on either the leading- or lagging-strand template. Using the lys2DeltaA746 assay system, we investigated changes in the accumulation of complex events at hotspots when the direction of replication was reversed in repair-compromised cells with either wildtype Poleta, a deletion of Poleta, or a mutant of Poleta that cannot interact with PCNA. Our results suggest that there is a polymerase hierarchy between Poleta and Polzeta in the bypass of certain lesions and that the interaction of Poleta with PCNA is needed for some, but not all, spontaneous lesion bypass.     

Xeroderma pigmentosum variant and error-prone DNA polymerases

Replicative DNA synthesis is a faithful event which requires undamaged DNA and high fidelity DNA polymerases. If unrepaired damage remains in the template DNA during replication, specialised low fidelity DNA polymerases synthesises DNA past lesions (translesion synthesis, TLS). Current evidence suggests that the polymerase switch from replicative to translesion polymerases might be mediated by post-translational modifications involving ubiquitination processes. One of these TLS polymerases, polymerase eta carries out TLS past UV photoproducts and is deficient in the variant form of xeroderma pigmentosum (XP-V). The dramatic proneness to skin cancer of XP-V individuals highlights the importance of this DNA polymerase in cancer avoidance. The UV hypermutability of XP-V cells suggests that, in the absence of a functional poleta, UV-induced lesions are bypassed by inaccurate DNA polymerase(s) which remain to be identified.     

Translesion Synthesis of Abasic Sites by Yeast DNA Polymerase ?

Studies of replicative DNA polymerases have led to the generalization that abasic sites are strong blocks to DNA replication. Here we show that yeast replicative DNA polymerase ϵ bypasses a model abasic site with comparable efficiency to Pol η and Dpo4, two translesion polymerases. DNA polymerase ϵ also exhibited high bypass efficiency with a natural abasic site on the template. Translesion synthesis primarily resulted in deletions. In cases where only a single nucleotide was inserted, dATP was the preferred nucleotide opposite the natural abasic site. In contrast to translesion polymerases, DNA polymerase ϵ with 3′–5′ proofreading exonuclease activity bypasses only the model abasic site during processive synthesis and cannot reinitiate DNA synthesis. This characteristic may allow other pathways to rescue leading strand synthesis when stalled at an abasic site.     

Requirement of Rad5 for DNA polymerase zeta-dependent translesion synthesis in Saccharomyces cerevisiae

In yeast, Rad6-Rad18-dependent lesion bypass involves translesion synthesis (TLS) by DNA polymerases eta or zeta or Rad5-dependent postreplication repair (PRR) in which error-free replication through the DNA lesion occurs by template switching. Rad5 functions in PRR via its two distinct activities-a ubiquitin ligase that promotes Mms2-Ubc13-mediated K63-linked polyubiquitination of PCNA at its lysine 164 residue and a DNA helicase that is specialized for replication fork regression. Both these activities are important for Rad5's ability to function in PRR. Here we provide evidence for the requirement of Rad5 in TLS mediated by Polzeta. Using duplex plasmids carrying different site-specific DNA lesions-an abasic site, a cis-syn TT dimer, a (6-4) TT photoproduct, or a G-AAF adduct-we show that Rad5 is needed for Polzeta-dependent TLS. Rad5 action in this role is likely to be structural, since neither the inactivation of its ubiquitin ligase activity nor the inactivation of its helicase activity impairs its role in TLS.     

Pyrosequencing for the quantitative assessment of 8-oxodG bypass DNA synthesis

Translesion synthesis (TLS) with specialized DNA polymerases allows dealing with a base lesion on the template strand during DNA replication; a base is inserted opposite the lesion, correctly or incorrectly, depending on the lesion, the involved DNA polymerase(s) and the sequence context. The major oxidized DNA base 8-oxo-7, 8-dihydro-2′-deoxyguanosine (8-oxodG) is highly mutagenic due to its ability to pair with either cytosine or adenine during DNA synthesis, depending on its conformation and involved DNA polymerases. To measure the correct or mutagenic outcome of lesion bypass, an original quantitative pyrosequencing method was developed and analytically validated. The method was applied to the study of DNA synthesis fidelity through an 8-oxodG or an undamaged guanine. After an in vitro primer-extension through 8-oxodG in the presence of the four deoxynucleotides triphosphates and a total nuclear protein extract, obtained from normal human intestinal epithelial cells (FHs 74 Int cell line), the reaction products were amplified by polymerase chain reaction and analyzed by pyrosequencing to measure nucleotides inserted opposite the lesion. The 8-oxodG bypass fidelity of FHs 74 Int cells nuclear extract is about 85.3%. We calculated within-day and total precisions for both 8-oxodG (2.8% and 2.8%, respectively) and undamaged templates (1.0% and 1.1%, respectively). We also demonstrated that only cytosine is incorporated opposite a normal guanine and that both cytosine and adenine can be incorporated opposite an 8-oxodG lesion. The proposed method is straightforward, fast, reproducible and easily adaptable to other sequences and lesions. It thus has a wide range of applications in the biological field, notably to elucidate TLS mechanisms and modulators.     

Analysis of CPD Ultraviolet Lesion Bypass in Chicken DT40 Cells: Polymerase η and PCNA Ubiquitylation Play Identical Roles

Translesion synthesis (TLS) provides a mechanism of copying damaged templates during DNA replication. This potentially mutagenic process may operate either at the replication fork or at post-replicative gaps. We used the example of T-T cyclobutane pyrimidine dimer (CPD) bypass to determine the influence of polymerase recruitment via PCNA ubiquitylation versus the REV1 protein on the efficiency and mutagenic outcome of TLS. Using mutant chicken DT40 cell lines we show that, on this numerically most important UV lesion, defects in polymerase η or in PCNA ubiquitylation similarly result in the long-term failure of lesion bypass with persistent strand gaps opposite the lesion, and the elevation of mutations amongst successful TLS events. Our data suggest that PCNA ubiquitylation promotes CPD bypass mainly by recruiting polymerase η, resulting in the majority of CPD lesions bypassed in an error-free manner. In contrast, we find that polymerase ζ is responsible for the majority of CPD-dependent mutations, but has no essential function in the completion of bypass. These findings point to a hierarchy of access of the different TLS polymerases to the lesion, suggesting a temporal order of their recruitment. The similarity of REV1 and REV3 mutant phenotypes confirms that the involvement of polymerase ζ in TLS is largely determined by its recruitment to DNA by REV1. Our data demonstrate the influence of the TLS polymerase recruitment mechanism on the success and accuracy of bypass.     

The effect of oxidative metabolism on spontaneous Pol zeta-dependent translesion synthesis in Saccharomyces cerevisiae

DNA lesions can stall or block high-fidelity polymerases, thus inhibiting replication. To bypass such lesions, low-fidelity translesion synthesis (TLS) polymerases can be used to insert a nucleotide across from the lesion or extend from a lesion:base mispair. When DNA repair is compromised in Saccharomyces cerevisiae, spontaneous DNA lesions can lead to a novel mutational event in which a frameshift is accompanied by one or more base pair substitutions. These "complex frameshifts" are dependent upon the TLS polymerase Pol zeta, and provide a mutational signature for mutagenic Pol zeta-dependent activity. In the current study, we have found that a specific subset of the Pol zeta-dependent mutational events requires oxidative metabolism. These results suggest that translesion bypass of spontaneously oxidized DNA bases can be a significant source of mutagenesis in repair compromised cells.     

Error-free and error-prone lesion bypass by human DNA polymerase kappa in vitro

Error-free lesion bypass and error-prone lesion bypass are important cellular responses to DNA damage during replication, both of which require a DNA polymerase (Pol). To identify lesion bypass DNA polymerases, we have purified human Polκ encoded by the DINB1 gene and examined its response to damaged DNA templates. Here, we show that human Polκ is a novel lesion bypass polymerase in vitro. Purified human Polκ efficiently bypassed a template 8-oxoguanine, incorporating mainly A and less frequently C opposite the lesion. Human Polκ most frequently incorporated A opposite a template abasic site. Efficient further extension required T as the next template base, and was mediated mainly by a one-nucleotide deletion mechanism. Human Polκ was able to bypass an acetylaminofluorene-modified G in DNA, incorporating either C or T, and less efficiently A opposite the lesion. Furthermore, human Polκ effectively bypassed a template (–)-trans-anti-benzo[a]pyrene-N²-dG lesion in an error-free manner by incorporating a C opposite the bulky adduct. In contrast, human Polκ was unable to bypass a template TT dimer or a TT (6-4) photoproduct, two of the major UV lesions. These results suggest that Polκ plays an important role in both error-free and error-prone lesion bypass in humans.     

Altered translesion synthesis in E. coli Pol V mutants selected for increased recombination inhibition

Replication of damaged DNA, also termed as translesion synthesis (TLS), involves specialized DNA polymerases that bypass DNA lesions. In Escherichia coli, although TLS can involve one or a combination of DNA polymerases depending on the nature of the lesion, it generally requires the Pol V DNA polymerase (formed by two SOS proteins, UmuD' and UmuC) and the RecA protein. In addition to being an essential component of translesion DNA synthesis, Pol V is also an antagonist of RecA-mediated recombination. We have recently isolated umuD' and umuC mutants on the basis of their increased capacity to inhibit homologous recombination. Despite the capacity of these mutants to form a Pol V complex and to interact with the RecA polymer, most of them exhibit a defect in TLS. Here, we further characterize the TLS activity of these Pol V mutants in vivo by measuring the extent of error-free and mutagenic bypass at a single (6-4)TT lesion located in double stranded plasmid DNA. TLS is markedly decreased in most Pol V mutants that we analyzed (8/9) with the exception of one UmuC mutant (F287L) that exhibits wild-type bypass activity. Somewhat unexpectedly, Pol V mutants that are partially deficient in TLS are more severely affected in mutagenic bypass compared to error-free synthesis. The defect in bypass activity of the Pol V mutant polymerases is discussed in light of the location of the respective mutations in the 3D structure of UmuD' and the DinB/UmuC homologous protein Dpo4 of Sulfolobus solfataricus.     

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.     

Translesion synthesis by human DNA polymerase eta across thymine glycol lesions

The XP-V (xeroderma pigmentosum variant) gene product, human DNA polymerase eta (pol eta), catalyzes efficient and accurate translesion synthesis (TLS) past cis-syn thymine-thymine dimers (TT dimer). In addition, recent reports suggest that pol eta is involved in TLS past various other types of lesion, including an oxidative DNA damage, 8-hydroxyguanine. Here, we compare the abilities of pol alpha and pol eta to replicate across thymine glycol (Tg) using purified synthetic oligomers containing a 5R- or 5S-Tg. DNA synthesis by pol alpha was inhibited at both steps of insertion of a nucleotide opposite the lesion and extension from the resulting product, indicating that pol alpha can weakly contribute to TLS past Tg lesions. In contrast, pol eta catalyzed insertion opposite the lesion as efficient as that opposite undamaged T, while extension was inhibited especially on the 5S-Tg template. Thus, pol eta catalyzed relatively efficient TLS past 5R-Tg than 5S-Tg. To compare the TLS abilities of pol eta for these lesions, we determined the kinetic parameters of pol eta for catalyzing TLS past a TT dimer, an N-2-acetylaminofluorene-modified guanine, and an abasic site analogue. The possible mechanisms of pol eta-catalyzed TLS are discussed on the basis of these results.     

Keeping Mammalian Mutation Load in Check: Regulation of the Activity of Error-Prone DNA Polymerases by p53 and p21

To overcome DNA lesions that block replication the cell employs translesion DNA synthesis (TLS) polymerases, a group of low fidelity DNA polymerases that have the capacity to bypass a wide range of DNA lesions. This TLS process is also termed error-prone repair, due to its inherent mutagenic nature. We have recently shown that the tumor suppressor p53 and the cell cycle inhibitor p21 are global regulators of TLS. When these proteins are missing or non-functional, TLS gets out of control: its extent increases to very high levels, and its fidelity decreases, causing an overall increase in mutation load. This may be explained by the loss of selectivity in the bypass of specific DNA lesions by their cognate specialized polymerases, such that lesion bypass continues to a maximum, regardless of the price paid in increased mutations. The p53 and p21 proteins are also required for efficient UV light-induced monoubiquitination of PCNA, which is consistent with a model in which this modification of PCNA is necessary but not sufficient for the normal activity of TLS. This regulation suggests that TLS evolved in mammals as a system that balances gain in survival with a tolerable mutational cost, and that disturbing this balance causes a potentially harmful increase in mutations, which might play a role in carcinogenesis.     

Loss of DNA minor groove interactions by exonuclease-deficient Klenow polymerase inhibits O6-methylguanine and abasic site translesion synthesis

The importance of DNA polymerase-DNA minor groove interactions on translesion synthesis (TLS) was examined in vitro using variants of exonuclease-deficient Klenow polymerase and site-specifically modified DNA oligonucleotides. Polymerase variant R668A lacks primer strand interactions, while variant Q849A lacks template strand interactions. O(6)-Methylguanine (m6G) and abasic site TLS was examined in three stages: dNTP insertion opposite the lesion, extension from a terminal lesion-containing base pair, and the dissociation equilibrium of the polymerase from the lesion-containing template. Less than 5% TLS was observed at the insertion step for either variant on the lesion-containing templates. While extensive TLS was observed for WT polymerase on the m6G template, only incorporation opposite the lesion was observed for the R668A variant. Loss of the template strand interaction, Q849A, resulted in the inability to insert dNTPs opposite either the m6G or abasic lesion. For both variants, extension of purine-containing m6G primer-templates was increased relative to WT polymerase. We observed similar extension efficiencies for all variants, relative to WT, using abasic template-primers. Polymerase dissociation/reassociation was studied through the use of a competitor primer/template complex. Dissociation for WT polymerase increased 2-fold and 3-fold, respectively, for m6G and abasic lesion-containing templates, relative to the natural template. Variants lacking DNA minor groove interactions displayed increased dissociation from DNA templates, relative to WT polymerase, but do not display an increased level of lesion-induced polymerase dissociation. Our results indicate that the primer and template strand interactions of the Klenow polymerase with the DNA minor groove are critical for maintaining the DNA-polymerase complex during translesion synthesis.     

The essential C family DnaE polymerase is error-prone and efficient at lesion bypass

DnaE-type DNA polymerases belong to the C family of DNA polymerases and are responsible for chromosomal replication in prokaryotes. Like most closely related Gram-positive cells, Streptococcus pyogenes has two DnaE homologs Pol C and DnaE; both are essential to cell viability. Pol C is an established replicative polymerase, and DnaE has been proposed to serve a replicative role. In this report, we characterize S. pyogenes DnaE polymerase and find that it is highly error-prone. DnaE can bypass coding and noncoding lesions with high efficiency. Error-prone extension is accomplished by either of two pathways, template-primer misalignment or direct primer extension. The bypass of abasic sites is accomplished mainly through "dNTP-stabilized" misalignment of template, thereby generating (-1) deletions in the newly synthesized strand. This mechanism may be similar to the dNTP-stabilized misalignment mechanism used by the Y family of DNA polymerases and is the first example of lesion bypass and error-prone synthesis catalyzed by a C family polymerase. Thus, DnaE may function in an error-prone capacity that may be essential in Gram-positive cells but not Gram-negative cells, suggesting a fundamental difference in DNA metabolism between these two classes of bacteria.     

Quantitative analysis of the efficiency and mutagenic spectra of abasic lesion bypass catalyzed by human Y-family DNA polymerases

Higher eukaryotes encode various Y-family DNA polymerases to perform global DNA lesion bypass. To provide complete mutation spectra for abasic lesion bypass, we employed short oligonucleotide sequencing assays to determine the sequences of abasic lesion bypass products synthesized by human Y-family DNA polymerases eta (hPolη), iota (hPolι) and kappa (hPolκ). The fourth human Y-family DNA polymerase, Rev1, failed to generate full-length lesion bypass products after 3 h. The results indicate that hPolι generates mutations with a frequency from 10 to 80% during each nucleotide incorporation event. In contrast, hPolη is the least error prone, generating the fewest mutations in the vicinity of the abasic lesion and inserting dAMP with a frequency of 67% opposite the abasic site. While the error frequency of hPolκ is intermediate to those of hPolη and hPolι, hPolκ has the highest potential to create frameshift mutations opposite the abasic site. Moreover, the time (t₅₀^bypass) required to bypass 50% of the abasic lesions encountered by hPolη, hPolι and hPolκ was 4.6, 112 and 1 823 s, respectively. These t₅₀^bypass values indicate that, among the enzymes, hPolη has the highest abasic lesion bypass efficiency. Together, our data suggest that hPolη is best suited to perform abasic lesion bypass in vivo.