RPA and PCNA suppress formation of large deletion errors by yeast DNA polymerase delta

In fulfilling its biosynthetic roles in nuclear replication and in several types of repair, DNA polymerase δ (pol δ) is assisted by replication protein A (RPA), the single-stranded DNA-binding protein complex, and by the processivity clamp proliferating cell nuclear antigen (PCNA). Here we report the effects of these accessory proteins on the fidelity of DNA synthesis in vitro by yeast pol δ. We show that when RPA and PCNA are included in reactions containing pol δ, rates for single base errors are similar to those generated by pol δ alone, indicating that pol δ itself is by far the prime determinant of fidelity for single base errors. However, the rate of deleting multiple nucleotides between directly repeated sequences is reduced by ~10-fold in the presence of either RPA or PCNA, and by ≥90-fold when both proteins are present. We suggest that PCNA and RPA suppress large deletion errors by preventing the primer terminus at a repeat from fraying and/or from relocating and annealing to a downstream repeat. Strong suppression of deletions by PCNA and RPA suggests that they may contribute to the high replication fidelity needed to stably maintain eukaryotic genomes that contain abundant repetitive sequences.     

In vitro gap-directed translesion DNA synthesis of an abasic site involving human DNA polymerases epsilon, lambda, and beta

DNA polymerase (pol) ε is thought to be the leading strand replicase in eukaryotes, whereas pols λ and β are believed to be mainly involved in re-synthesis steps of DNA repair. DNA elongation by the human pol ε is halted by an abasic site (apurinic/apyrimidinic (AP) site). In this study, we present in vitro evidence that human pols λ, β, and η can perform translesion synthesis (TLS) of an AP site in the presence of pol ε, likely by initiating the 3'OHs created at the lesion by the arrested pol ε. However, in the case of pols λ and β, this TLS requires the presence of a DNA gap downstream from the product synthesized by the pol ε, and the optimal gap for efficient TLS is different for the two polymerases. The presence of gaps did not affect the TLS capacity of human pol η. Characterization of the reaction products showed that pol β inserted dAMP opposite the AP site, whereas gap filling synthesis by pol λ resulted in single or double deletions opposite the lesion. The synthesis up to the AP site by pol ε and the subsequent TLS by pols λ and β are not influenced by human processivity factor proliferating cell nuclear antigen and human single-stranded DNA-binding protein replication protein A. The bypass capacity of pol λ at the AP site is greatly reduced when a truncated form of the enzyme, which has lost the BRCA1 C-terminal and proline-rich domains, is used. Collectively, our in vitro results support the existence of a mechanism of gap-directed TLS at an AP site involving a switch between the replicative pol ε and the repair pols λ and β.     

Error-free bypass of 2-hydroxyadenine by human DNA polymerase lambda with Proliferating Cell Nuclear Antigen and Replication Protein A in different sequence contexts

1,2-dihydro-2-oxoadenine (2-OH-A), a common DNA lesion produced by reactive oxygen species, is a strong replicative block for several DNA polymerases (DNA pols). We have previously shown that various bases can be misincorporated opposite the 2-OH-A lesion and the type of mispairs varies with either the sequence context or the type of DNA pol tested. Here, we have analysed the ability of the human pol family X member DNA pol λ, to bypass the 2-OH-A lesion. DNA pol λ can perform error-free bypass of 2-OH-A when this lesion is located in a random sequence, whereas in a repeated sequence context, even though bypass was also largely error-free, misincorporation of dGMP could be observed. The fidelity of translesion synthesis of 2-OH-A in a repeated sequence by DNA pol λ was enhanced by the auxiliary proteins Proliferating Cell Nuclear Antigen (PCNA) and Replication Protein A (RP-A). We also found that the DNA pol λ active site residue tyrosine 505 determined the nucleotide selectivity opposite 2-OH-A. Our data show, for the first time, that the 2-OH-A lesion can be efficiently and faithfully bypassed by a human DNA pol λ in combination with PCNA and RP-A.     

The Block of DNA Polymerase �� Strand Displacement Activity by an Abasic Site Can Be Rescued by the Concerted Action of DNA Polymerase �� and Flap Endonuclease 1

Abasic (AP) sites are very frequent and dangerous DNA lesions. Their ability to block the advancement of a replication fork has been always viewed as a consequence of their inhibitory effect on the DNA synthetic activity of replicative DNA polymerases (DNA pols). Here we show that AP sites can also affect the strand displacement activity of the lagging strand DNA pol δ, thus preventing proper Okazaki fragment maturation. This block can be overcome through a polymerase switch, involving the combined physical and functional interaction of DNA pol β and Flap endonuclease 1. Our data identify a previously unnoticed deleterious effect of the AP site lesion on normal cell metabolism and suggest the existence of a novel repair pathway that might be important in preventing replication fork stalling.Loss of purine and pyrimidine bases is a significant source of DNA damage in prokaryotic and eukaryotic organisms. Abasic (apurinic and apyrimidinic) lesions occur spontaneously in DNA; in eukaryotes it has been estimated that about 10⁴ depurination and 10² depyrimidation events occur per genome per day. An equally important source of abasic DNA lesions results from the action of DNA glycosylases, such as uracil glycosylase, which excises uracil arising primarily from spontaneous deamination of cytosines (1). Although most AP sites are removed by the base excision repair (BER)⁵ pathway, a small fraction of lesions persists, and DNA with AP lesions presents a strong block to DNA synthesis by replicative DNA polymerases (DNA pols) (2, 3). Several studies have been performed to address the effects of AP sites on the template DNA strand on the synthetic activity of a variety of DNA pols. The major replicative enzyme of eukaryotic cells, DNA pol δ, was shown to be able to bypass an AP lesion, but only in the presence of the auxiliary factor proliferating cell nuclear antigen (PCNA) and at a very reduced catalytic efficiency if compared with an undamaged DNA template (4). On the other hand, the family X DNA pols β and λ were shown to bypass an AP site but in a very mutagenic way (5). Recent genetic evidence in Saccharomyces cerevisiae cells showed that DNA pol δ is the enzyme replicating the lagging strand (6). According to the current model for Okazaki fragment synthesis (7–9), the action of DNA pol δ is not only critical for the extension of the newly synthesized Okazaki fragment but also for the displacement of an RNA/DNA segment of about 30 nucleotides on the pre-existing downstream Okazaki fragment to create an intermediate Flap structure that is the target for the subsequent action of the Dna2 endonuclease and the Flap endonuclease 1 (Fen-1). This process has the advantage of removing the entire RNA/DNA hybrid fragment synthesized by the DNA pol α/primase, potentially containing nucleotide misincorporations caused by the lack of a proofreading exonuclease activity of DNA pol α/primase. This results in a more accurate copy synthesized by DNA pol δ. The intrinsic strand displacement activity of DNA pol δ, in conjunction with Fen-1, PCNA, and replication protein A (RP-A), has been also proposed to be essential for the S phase-specific long patch BER pathway (10, 11). Although it is clear that an AP site on the template strand is a strong block for DNA pol δ-dependent synthesis on single-stranded DNA, the functional consequences of such a lesion on the ability of DNA pol δ to carry on strand displacement synthesis have never been investigated so far. Given the high frequency of spontaneous hydrolysis and/or cytidine deamination events, any detrimental effect of an AP site on the strand displacement activity of DNA pol δ might have important consequences both for lagging strand DNA synthesis and for long patch BER. In this work, we addressed this issue by constructing a series of synthetic gapped DNA templates with a single AP site at different positions with respect to the downstream primer to be displaced by DNA pol δ (see Fig. 1A). We show that an AP site immediately upstream of a single- to double-strand DNA junction constitutes a strong block to the strand displacement activity of DNA pol δ, even in the presence of RP-A and PCNA. Such a block could be resolved only through a “polymerase switch” involving the concerted physical and functional interaction of DNA pol β and Fen-1. The closely related DNA pol λ could only partially substitute for DNA pol β. Based on our data, we propose that stalling of a replication fork by an AP site not only is a consequence of its ability to inhibit nucleotide incorporation by the replicative DNA pols but can also stem from its effects on strand displacement during Okazaki fragment maturation. In summary, our data suggest the existence of a novel repair pathway that might be important in preventing replication fork stalling and identify a previously unnoticed deleterious effect of the AP site lesion on normal cell metabolism.Open in a separate windowFIGURE 1.An abasic site immediately upstream of a double-stranded DNA region inhibits the strand displacement activity of DNA polymerase δ. The reactions were performed as described under “Experimental Procedures.” A, schematic representation of the various DNA templates used. The size of the resulting gaps is indicated in nt. The position of the AP site on the 100-mer template strand is indicated relative to the 3′ end. Base pairs in the vicinity of the lesion are indicated by dashes. The size of the gaps (35–38 nt) is consistent with the size of ssDNA covered by a single RP-A molecule, which has to be released during Okazaki fragment synthesis when the DNA pol is approaching the 5′-end of the downstream fragment. When the AP site is covered by the downstream terminator oligonucleotide (Gap-3 and Gap-1 templates) the nucleotide placed on the opposite strand is C to mimic the situation generated by spontaneous loss of a guanine or excision of an oxidized guanine, whereas when the AP site is covered by the primer (nicked AP template), the nucleotide placed on the opposite strand is A to mimic the most frequent incorporation event occurring opposite an AP site. B, human PCNA was titrated in the presence of 15 nm (lanes 2–4 and 10–12) or 30 nm (lanes 6–8 and 14–16) recombinant human four subunit DNA pol δ, on a linear control (lanes 1–8) or a 38-nt gap control (lanes 9–16) template. Lanes 1, 5, 9, and 13, control reactions in the absence of PCNA. C, human PCNA was titrated in the presence of 60 nm DNA pol δ, on a linear AP (lanes 2–4) or 38-nt gap AP (lanes 6–9) template. Lanes 1 and 5, control reactions in the absence of PCNA.     

Dynamics of human replication factors in the elongation phase of DNA replication

In eukaryotic cells, DNA replication is carried out by coordinated actions of many proteins, including DNA polymerase δ (pol δ), replication factor C (RFC), proliferating cell nuclear antigen (PCNA) and replication protein A. Here we describe dynamic properties of these proteins in the elongation step on a single-stranded M13 template, providing evidence that pol δ has a distributive nature over the 7 kb of the M13 template, repeating a frequent dissociation–association cycle at growing 3′-hydroxyl ends. Some PCNA could remain at the primer terminus during this cycle, while the remainder slides out of the primer terminus or is unloaded once pol δ has dissociated. RFC remains around the primer terminus through the elongation phase, and could probably hold PCNA from which pol δ has detached, or reload PCNA from solution to restart DNA synthesis. Furthermore, we suggest that a subunit of pol δ, POLD3, plays a crucial role in the efficient recycling of PCNA during dissociation–association cycles of pol δ. Based on these observations, we propose a model for dynamic processes in elongation complexes.     

Tracking of progressing human DNA polymerase δ holoenzymes reveals distributions of DNA lesion bypass activities

During DNA replication, DNA lesions in lagging strand templates are initially encountered by DNA polymerase δ (pol δ) holoenzymes comprised of pol δ and the PCNA processivity sliding clamp. These encounters are thought to stall replication of an afflicted template before the lesion, activating DNA damage tolerance (DDT) pathways that replicate the lesion and adjacent DNA sequence, allowing pol δ to resume. However, qualitative studies observed that human pol δ can replicate various DNA lesions, albeit with unknown proficiencies, which raises issues regarding the role of DDT in replicating DNA lesions. To address these issues, we re-constituted human lagging strand replication to quantitatively characterize initial encounters of pol δ holoenzymes with DNA lesions. The results indicate pol δ holoenzymes support dNTP incorporation opposite and beyond multiple lesions and the extent of these activities depends on the lesion and pol δ proofreading. Furthermore, after encountering a given DNA lesion, subsequent dissociation of pol δ is distributed around the lesion and a portion does not dissociate. The distributions of these events are dependent on the lesion and pol δ proofreading. Collectively, these results reveal complexity and heterogeneity in the replication of lagging strand DNA lesions, significantly advancing our understanding of human DDT.     

The fidelity of DNA synthesis by yeast DNA polymerase zeta alone and with accessory proteins

DNA polymerase zeta (pol ζ) participates in several DNA transactions in eukaryotic cells that increase spontaneous and damage-induced mutagenesis. To better understand this central role in mutagenesis in vivo, here we report the fidelity of DNA synthesis in vitro by yeast pol ζ alone and with RFC, PCNA and RPA. Overall, the accessory proteins have little effect on the fidelity of pol ζ. Pol ζ is relatively accurate for single base insertion/deletion errors. However, the average base substitution fidelity of pol ζ is substantially lower than that of homologous B family pols α, δ and . Pol ζ is particularly error prone for substitutions in specific sequence contexts and generates multiple single base errors clustered in short patches at a rate that is unprecedented in comparison with other polymerases. The unique error specificity of pol ζ in vitro is consistent with Pol ζ-dependent mutagenic specificity reported in vivo. This fact, combined with the high rate of single base substitution errors and complex mutations observed here, indicates that pol ζ contributes to mutagenesis in vivo not only by extending mismatches made by other polymerases, but also by directly generating its own mismatches and then extending them.     

The eukaryotic replisome tolerates leading‐strand base damage by replicase switching

The high‐fidelity replicative DNA polymerases, Pol ε and Pol δ, are generally thought to be poorly equipped to replicate damaged DNA. Direct and complete replication of a damaged template therefore typically requires the activity of low‐fidelity translesion synthesis (TLS) polymerases. Here we show that a yeast replisome, reconstituted with purified proteins, is inherently tolerant of the common oxidative lesion thymine glycol (Tg). Surprisingly, leading‐strand Tg was bypassed efficiently in the presence and absence of the TLS machinery. Our data reveal that following helicase–polymerase uncoupling a switch from Pol ε, the canonical leading‐strand replicase, to the lagging‐strand replicase Pol δ, facilitates rapid, efficient and error‐free lesion bypass at physiological nucleotide levels. This replicase switch mechanism also promotes bypass of the unrelated oxidative lesion, 8‐oxoguanine. We propose that replicase switching may promote continued leading‐strand synthesis whenever the replisome encounters leading‐strand damage that is bypassed more efficiently by Pol δ than by Pol ε.     

Biochemical Analysis of DNA Polymerase η Fidelity in the Presence of Replication Protein A

DNA polymerase η (pol η) synthesizes across from damaged DNA templates in order to prevent deleterious consequences like replication fork collapse and double-strand breaks. This process, termed translesion synthesis (TLS), is an overall positive for the cell, as cells deficient in pol η display higher mutation rates. This outcome occurs despite the fact that the in vitro fidelity of bypass by pol η alone is moderate to low, depending on the lesion being copied. One possible means of increasing the fidelity of pol η is interaction with replication accessory proteins present at the replication fork. We have previously utilized a bacteriophage based screening system to measure the fidelity of bypass using purified proteins. Here we report on the fidelity effects of a single stranded binding protein, replication protein A (RPA), when copying the oxidative lesion 7,8-dihydro-8-oxo-guanine(8-oxoG) and the UV-induced cis-syn thymine-thymine cyclobutane pyrimidine dimer (T-T CPD). We observed no change in fidelity dependent on RPA when copying these damaged templates. This result is consistent in multiple position contexts. We previously identified single amino acid substitution mutants of pol η that have specific effects on fidelity when copying both damaged and undamaged templates. In order to confirm our results, we examined the Q38A and Y52E mutants in the same full-length construct. We again observed no difference when RPA was added to the bypass reaction, with the mutant forms of pol η displaying similar fidelity regardless of RPA status. We do, however, observe some slight effects when copying undamaged DNA, similar to those we have described previously. Our results indicate that RPA by itself does not affect pol η dependent lesion bypass fidelity when copying either 8-oxoG or T-T CPD lesions.     

Basis of Miscoding of the DNA Adduct N2,3-Ethenoguanine by Human Y-family DNA Polymerases

N²,3-Ethenoguanine (N²,3-ϵG) is one of the exocyclic DNA adducts produced by endogenous processes (e.g. lipid peroxidation) and exposure to bioactivated vinyl monomers such as vinyl chloride, which is a known human carcinogen. Existing studies exploring the miscoding potential of this lesion are quite indirect because of the lability of the glycosidic bond. We utilized a 2′-fluoro isostere approach to stabilize this lesion and synthesized oligonucleotides containing 2′-fluoro-N²,3-ϵ-2′-deoxyarabinoguanosine to investigate the miscoding potential of N²,3-ϵG by Y-family human DNA polymerases (pols). In primer extension assays, pol η and pol κ replicated through N²,3-ϵG, whereas pol ι and REV1 yielded only 1-base incorporation. Steady-state kinetics revealed that dCTP incorporation is preferred opposite N²,3-ϵG with relative efficiencies in the order of pol κ > REV1 > pol η ≈ pol ι, and dTTP misincorporation is the major miscoding event by all four Y-family human DNA pols. Pol ι had the highest dTTP misincorporation frequency (0.71) followed by pol η (0.63). REV1 misincorporated dTTP and dGTP with much lower frequencies. Crystal structures of pol ι with N²,3-ϵG paired to dCTP and dTTP revealed Hoogsteen-like base pairing mechanisms. Two hydrogen bonds were observed in the N²,3-ϵG:dCTP base pair, whereas only one appears to be present in the case of the N²,3-ϵG:dTTP pair. Base pairing mechanisms derived from the crystal structures explain the slightly favored dCTP insertion for pol ι in steady-state kinetic analysis. Taken together, these results provide a basis for the mutagenic potential of N²,3-ϵG.     

Roles of PCNA ubiquitination and TLS polymerases κ and η in the bypass of methyl methanesulfonate-induced DNA damage

Translesion synthesis (TLS) provides a highly conserved mechanism that enables DNA synthesis on a damaged template. TLS is performed by specialized DNA polymerases of which polymerase (Pol) κ is important for the cellular response to DNA damage induced by benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), ultraviolet (UV) light and the alkylating agent methyl methanesulfonate (MMS). As TLS polymerases are intrinsically error-prone, tight regulation of their activity is required. One level of control is provided by ubiquitination of the homotrimeric DNA clamp PCNA at lysine residue 164 (PCNA-Ub). We here show that Polκ can function independently of PCNA modification and that Polη can function as a backup during TLS of MMS-induced lesions. Compared to cell lines deficient for PCNA modification (Pcna^K164R) or Polκ, double mutant cell lines display hypersensitivity to MMS but not to BPDE or UV-C. Double mutant cells also displayed delayed post-replicative TLS, accumulate higher levels of replication stress and delayed S-phase progression. Furthermore, we show that Polη and Polκ are redundant in the DNA damage bypass of MMS-induced DNA damage. Taken together, we provide evidence for PCNA-Ub-independent activation of Polκ and establish Polη as an important backup polymerase in the absence of Polκ in response to MMS-induced DNA damage.     

A Role for DNA Polymerase θ in Promoting Replication through Oxidative DNA Lesion,Thymine Glycol,in Human Cells

The biological functions of human DNA polymerase (pol) θ, an A family polymerase, have remained poorly defined. Here we identify a role of polθ in translesion synthesis (TLS) in human cells. We show that TLS through the thymine glycol (TG) lesion, the most common oxidation product of thymine, occurs via two alternative pathways, in one of which, polymerases κ and ζ function together and mediate error-free TLS, whereas in the other, polθ functions in an error-prone manner. Human polθ is comprised of an N-terminal ATPase/helicase domain, a large central domain, and a C-terminal polymerase domain; however, we find that only the C-terminal polymerase domain is required for TLS opposite TG in human cells. In contrast to TLS mediated by polκ and polζ, in which polζ would elongate the chain from the TG:A base pair formed by polκ action, the ability of polθ alone to carry out the nucleotide insertion step, as well as the subsequent extension step that presents a considerable impediment due to displacement of the 5′ template base, suggests that the polθ active site can accommodate highly distorting DNA lesions.     

Human replication protein A can suppress the intrinsic in vitro mutator phenotype of human DNA polymerase lambda

DNA polymerase λ (pol λ) is a member of the X family DNA polymerases and is endowed with multiple enzymatic activities. In this work we investigated the in vitro miscoding properties of full-length, human pol λ either in the absence or in the presence of the human auxiliary proteins proliferating cell nuclear antigen (PCNA) and replication protein A (RP-A). Our data suggested that (i) pol λ had an intrinsic ability to create mismatches and to incorporate ribonucleotides at nearly physiological Mn⁺⁺ and Mg⁺⁺ concentrations; (ii) the sequence of the template-primer could influence the misincorporation frequency of pol λ; (iii) pol λ preferentially generated G:T and G:G mismatches; (iv) RP-A, but not PCNA, selectively prevented misincorporation of an incorrect nucleotide by pol λ, without affecting correct incorporation and (v) this inhibitory effect required a precise ratio between the concentrations of pol λ and RP-A. Possible physiological implications of these findings for the in vivo fidelity of pol λ are discussed.     

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.     

The eukaryotic leading and lagging strand DNA polymerases are loaded onto primer-ends via separate mechanisms but have comparable processivity in the presence of PCNA

Saccharomyces cerevisiae DNA polymerase δ (Pol δ) and DNA polymerase ε (Pol ε) are replicative DNA polymerases at the replication fork. Both enzymes are stimulated by PCNA, although to different levels. To understand why and to explore the interaction with PCNA, we compared Pol δ and Pol ε in physical interactions with PCNA and nucleic acids (with or without RPA), and in functional assays measuring activity and processivity. Using surface plasmon resonance technique, we show that Pol ε has a high affinity for DNA, but a low affinity for PCNA. In contrast, Pol δ has a low affinity for DNA and a high affinity for PCNA. The true processivity of Pol δ and Pol ε was measured for the first time in the presence of RPA, PCNA and RFC on single-stranded DNA. Remarkably, in the presence of PCNA, the processivity of Pol δ and Pol ε on RPA-coated DNA is comparable. Finally, more PCNA molecules were found on the template after it was replicated by Pol ε when compared to Pol δ. We conclude that Pol ε and Pol δ exhibit comparable processivity, but are loaded on the primer-end via different mechanisms.     

Mutagenesis of human DNA polymerase lambda: essential roles of Tyr505 and Phe506 for both DNA polymerase and terminal transferase activities

DNA polymerase (pol) λ is homologous to pol β and has intrinsic polymerase and terminal transferase activities. However, nothing is known about the amino acid residues involved in these activites. In order to precisely define the nucleotide-binding site of human pol λ, we have mutagenised two amino acids, Tyr505 and the neighbouring Phe506, which were predicted by structural homology modelling to correspond to the Tyr271 and Phe272 residues of pol β, which are involved in nucleotide binding. Our analysis demonstrated that pol λ Phe506Arg/Gly mutants possess very low polymerase and terminal transferase activities as well as greatly reduced abilities for processive DNA synthesis and for carrying on translesion synthesis past an abasic site. The Tyr505Ala mutant, on the other hand, showed an altered nucleotide binding selectivity to perform the terminal transferase activity. Our results suggest the existence of a common nucleotide-binding site for the polymerase and terminal transferase activities of pol λ, as well as distinct roles of the amino acids Tyr505 and Phe506 in these two catalytic functions.     

Hymenoic acid, a novel specific inhibitor of human DNA polymerase lambda from a fungus of Hymenochaetaceae sp

Hymenoic acid (1) is a natural compound isolated from cultures of a fungus, Hymenochaetaceae sp., and this structure was determined by spectroscopic analyses. Compound 1 is a novel sesquiterpene, trans-4-[(1′E,5′S)-5′-carboxy-1′-methyl-1′-hexenyl]cyclohexanecarboxylic acid. This compound selectively inhibited the activity of human DNA polymerase λ (pol λ) in vitro, and 50% inhibition was observed at a concentration of 91.7 μM. Compound 1 did not influence the activities of the other seven mammalian pols (i.e., pols , γ, δ, ε, η, ι, and κ), but also showed no effect even on the activity of pol β, which is thought to have a very similar three-dimensional structure to the pol β-like region of pol λ. This compound also did not inhibit the activities of prokaryotic pols and other DNA metabolic enzymes tested. These results suggested that compound 1 could be a selective inhibitor of eukaryotic pol λ. This compound had no inhibitory activities against two N-terminal truncated pol λ, del-1 pol λ (lacking nuclear localization signal (NLS), BRCA1 C-terminus (BRCT) domain [residues 133–575]), and del-2 pol λ (lacking NLS, BRCT, domain and proline-rich region [residues 245–575]). The compound 1-induced inhibition of intact pol λ activity was non-competitive with respect to both the DNA template-primer and the dNTP substrate. On the basis of these results, the pol λ inhibitory mechanism of compound 1 is discussed.     

Structural and Kinetic Analysis of Nucleoside Triphosphate Incorporation Opposite an Abasic Site by Human Translesion DNA Polymerase η

The most common lesion in DNA is an abasic site resulting from glycolytic cleavage of a base. In a number of cellular studies, abasic sites preferentially code for dATP insertion (the “A rule”). In some cases frameshifts are also common. X-ray structures with abasic sites in oligonucleotides have been reported for several microbial and human DNA polymerases (pols), e.g. Dpo4, RB69, KlenTaq, yeast pol ι, human (h) pol ι, and human pol β. We reported previously that hpol η is a major pol involved in abasic site bypass (Choi, J.-Y., Lim, S., Kim, E. J., Jo, A., and Guengerich, F. P. (2010 J. Mol. Biol. 404, 34–44). hpol η inserted all four dNTPs in steady-state and pre-steady-state assays, preferentially inserting A and G. In LC-MS analysis of primer-template pairs, A and G were inserted but little C or T was inserted. Frameshifts were observed when an appropriate pyrimidine was positioned 5′ to the abasic site in the template. In x-ray structures of hpol η with a non-hydrolyzable analog of dATP or dGTP opposite an abasic site, H-bonding was observed between the phosphate 5′ to the abasic site and water H-bonded to N1 and N6 of A and N1 and O6 of G nucleoside triphosphate analogs, offering an explanation for what appears to be a “purine rule.” A structure was also obtained for an A inserted and bonded in the primer opposite the abasic site, but it did not pair with a 5′ T in the template. We conclude that hpol η, a major copying enzyme with abasic sites, follows a purine rule, which can also lead to frameshifts. The phenomenon can be explained with H-bonds.