Enhancement of Human DNA Polymerase η Activity and Fidelity Is Dependent Upon a Bipartite Interaction with the Werner Syndrome Protein

We have investigated the interaction between human DNA polymerase η (hpol η) and the Werner syndrome protein (WRN). Functional assays revealed that the WRN exonuclease and RecQ C-terminal (RQC) domains are necessary for full stimulation of hpol η-catalyzed formation of correct base pairs. We find that WRN does not stimulate hpol η-catalyzed formation of mispairs. Moreover, the exonuclease activity of WRN prevents stable mispair formation by hpol η. These results are consistent with a proofreading activity for WRN during single-nucleotide additions. ATP hydrolysis by WRN appears to attenuate stimulation of hpol η. Pre-steady-state kinetic results show that k_pol is increased 4-fold by WRN. Finally, pulldown assays reveal a bipartite physical interaction between hpol η and WRN that is mediated by the exonuclease and RQC domains. Taken together, these results are consistent with alteration of the rate-limiting step in polymerase catalysis by direct protein-protein interactions between WRN and hpol η. In summary, WRN improves the efficiency and fidelity of hpol η to promote more effective replication of DNA.     

Kinetics,Structure, and Mechanism of 8-Oxo-7,8-dihydro-2′-deoxyguanosine Bypass by Human DNA Polymerase η

DNA damage incurred by a multitude of endogenous and exogenous factors constitutes an inevitable challenge for the replication machinery. Cells rely on various mechanisms to either remove lesions or bypass them in a more or less error-prone fashion. The latter pathway involves the Y-family polymerases that catalyze trans-lesion synthesis across sites of damaged DNA. 7,8-Dihydro-8-oxo-2′-deoxyguanosine (8-oxoG) is a major lesion that is a consequence of oxidative stress and is associated with cancer, aging, hepatitis, and infertility. We have used steady-state and transient-state kinetics in conjunction with mass spectrometry to analyze in vitro bypass of 8-oxoG by human DNA polymerase η (hpol η). Unlike the high fidelity polymerases that show preferential insertion of A opposite 8-oxoG, hpol η is capable of bypassing 8-oxoG in a mostly error-free fashion, thus preventing GC→AT transversion mutations. Crystal structures of ternary hpol η-DNA complexes and incoming dCTP, dATP, or dGTP opposite 8-oxoG reveal that an arginine from the finger domain assumes a key role in avoiding formation of the nascent 8-oxoG:A pair. That hpol η discriminates against dATP exclusively at the insertion stage is confirmed by structures of ternary complexes that allow visualization of the extension step. These structures with G:dCTP following either 8-oxoG:C or 8-oxoG:A pairs exhibit virtually identical active site conformations. Our combined data provide a detailed understanding of hpol η bypass of the most common oxidative DNA lesion.     

Translesion Synthesis across 1,N6-(2-Hydroxy-3-hydroxymethylpropan-1,3-diyl)-2′-deoxyadenosine (1,N6-γ-HMHP-dA) Adducts by Human and Archebacterial DNA Polymerases

The 1,N⁶-(2-Hydroxy-3-hydroxymethylpropan-1,3-diyl)-2′-deoxyadenosine (1,N⁶-γ-HMHP-dA) adducts are formed upon bifunctional alkylation of adenine nucleobases in DNA by 1,2,3,4-diepoxybutane, the putative ultimate carcinogenic metabolite of 1,3-butadiene. The presence of a substituted 1,N⁶-propano group on 1,N⁶-γ-HMHP-dA is expected to block the Watson-Crick base pairing of the adducted adenine with thymine, potentially contributing to mutagenesis. In this study, the enzymology of replication past site-specific 1,N⁶-γ-HMHP-dA lesions in the presence of human DNA polymerases (hpols) β, η, κ, and ι and archebacterial polymerase Dpo4 was investigated. Run-on gel analysis with all four dNTPs revealed that hpol η, κ, and Dpo4 were able to copy the modified template. In contrast, hpol ι inserted a single base opposite 1,N⁶-γ-HMHP-dA but was unable to extend beyond the damaged site, and a complete replication block was observed with hpol β. Single nucleotide incorporation experiments indicated that although hpol η, κ, and Dpo4 incorporated the correct nucleotide (dTMP) opposite the lesion, dGMP and dAMP were inserted with a comparable frequency. HPLC-ESI-MS/MS analysis of primer extension products confirmed the ability of bypass polymerases to insert dTMP, dAMP, or dGMP opposite 1,N⁶-γ-HMHP-dA and detected large amounts of −1 and −2 deletion products. Taken together, these results indicate that hpol η and κ enzymes bypass 1,N⁶-γ-HMHP-dA lesions in an error-prone fashion, potentially contributing to A→T and A→C transversions and frameshift mutations observed in cells following treatment with 1,2,3,4-diepoxybutane.     

Futile short-patch DNA base excision repair of adenine:8-oxoguanine mispair

8-Oxo-7, 8-dihydrodeoxyguanosine (8-oxo-dG), one of the representative oxidative DNA lesions, frequently mispairs with the incoming dAMP during mammalian DNA replication. Mispaired dA is removed by post-replicative base excision repair (BER) initiated by adenine DNA glycosylase, MYH, creating an apurinic (AP) site. The subsequent mechanism ensuring a dC:8-oxo-dG pair, a substrate for 8-oxoguanine DNA glycosylase (OGG1), remains to be elucidated. At the nucleotide insertion step, none of the mammalian DNA polymerases examined exclusively inserted dC opposite 8-oxo-dG that was located in a gap. AP endonuclease 1, which possesses 3′→5′ exonuclease activity and potentially serves as a proofreader, did not discriminate dA from dC that was located opposite 8-oxo-dG. However, human DNA ligases I and III joined 3′-dA terminus much more efficiently than 3′-dC terminus when paired to 8-oxo-dG. In reconstituted short-patch BER, repair products contained only dA opposite 8-oxo-dG. These results indicate that human DNA ligases discriminate dC from dA and that MYH-initiated short-patch BER is futile and hence this BER must proceed to long-patch repair, even if it is initiated as short-patch repair, through strand displacement synthesis from the ligation-resistant dC terminus to generate the OGG1 substrate, dC:8-oxo-dG pair.     

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.     

A highly conserved Tyrosine residue of family B DNA polymerases contributes to dictate translesion synthesis past 8-oxo-7,8-dihydro-2'-deoxyguanosine

The harmfulness of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8oxodG) damage resides on its dual coding potential, as it can pair with the correct dCMP (dC) or the incorrect dAMP (dA). Here, we investigate the translesional synthesis ability of family B ϕ29 DNA polymerase on 8oxodG-containing templates. We show that this polymerase preferentially inserts dC opposite 8oxodG, its 3′–5′ exonuclease activity acting indistinctly on both dA or dC primer terminus. In addition, ϕ29 DNA polymerase shows a favoured extension of the 8oxodG/dA pair, but with an efficiency much lower than that of the canonical dG/dC pair. Additionally, we have analysed the role of the invariant tyrosine from motif B of family B DNA polymerases in translesional synthesis past 8oxodG, replacing the corresponding ϕ29 DNA polymerase Tyr390 by Phe or Ser. The lack of the aromatic portion in mutant Y390S led to a lost of discrimination against dA insertion opposite 8oxodG. On the contrary, the absence of the hydroxyl group in the Y390F mutant precluded the favoured extension of 8oxodG:dA base pair with respect to 8oxodG:dC. Based on the results obtained, we propose that this Tyr residue contributes to dictate nucleotide insertion and extension preferences during translesion synthesis past 8oxodG by family B replicases.     

Response of human REV1 to different DNA damage: preferential dCMP insertion opposite the lesion

REV1 functions in the DNA polymerase ζ mutagenesis pathway. To help understand the role of REV1 in lesion bypass, we have examined activities of purified human REV1 opposite various template bases and several different DNA lesions. Lacking a 3′→5′ proofreading exonuclease activity, purified human REV1 exhibited a DNA polymerase activity on a repeating template G sequence, but catalyzed nucleotide insertion with 6-fold lower efficiency opposite a template A and 19–27-fold lower efficiency opposite a template T or C. Furthermore, dCMP insertion was greatly preferred regardless of the specific template base. Human REV1 inserted a dCMP efficiently opposite a template 8-oxoguanine, (+)-trans-anti-benzo[a]pyrene-N ²-dG, (–)-trans-anti-benzo[a]pyrene-N ²-dG and 1,N ⁶-ethenoadenine adducts, very inefficiently opposite an acetylaminofluorene-adducted guanine, but was unresponsive to a template TT dimer or TT (6–4) photoproduct. Surprisingly, the REV1 specificity of nucleotide insertion was very similar in response to different DNA lesions with greatly preferred C insertion and least frequent A insertion. By combining the dCMP insertion activity of human REV1 with the extension synthesis activity of human polymerase κ, bypass of the trans-anti-benzo[a]pyrene-N ² -dG adducts and the 1,N ⁶-ethenoadenine lesion was achieved by the two-polymerase two-step mechanism. These results suggest that human REV1 is a specialized DNA polymerase that may contribute to dCMP insertion opposite many types of DNA damage during lesion bypass.     

Catalytic activities of Werner protein are affected by adduction with 4-hydroxy-2-nonenal

4-Hydroxy-2-nonenal (HNE) is a reactive α,β-unsaturated aldehyde generated during oxidative stress and subsequent peroxidation of polyunsaturated fatty acids. Here, Werner protein (WRN) was identified as a novel target for modification by HNE. Werner syndrome arises through mutations in the WRN gene that encodes the RecQ DNA helicase which is critical for maintaining genomic stability. This hereditary disease is associated with chromosomal instability, premature aging and cancer predisposition. WRN appears to participate in the cellular response to oxidative stress and cells devoid of WRN display elevated levels of oxidative DNA damage. We demonstrated that helicase/ATPase and exonuclease activities of HNE-modified WRN protein were inhibited both in vitro and in immunocomplexes purified from the cell extracts. Sites of HNE adduction in human WRN were identified at Lys577, Cys727, His1290, Cys1367, Lys1371 and Lys1389. We applied in silico modeling of the helicase and RQC domains of WRN protein with HNE adducted to Lys577 and Cys727 and provided a potential mechanism of the observed deregulation of the protein catalytic activities. In light of the obtained results, we postulate that HNE adduction to WRN is a post-translational modification, which may affect WRN conformational stability and function, contributing to features and diseases associated with premature senescence.     

168 NMR study of RQC domain of BLM protein

BLM, a member of the RecQ helicase associated with the Bloom’s syndrome human genetic disorder, has been found to bind to noncanonical DNA with high affinity via its RecQ C-terminal domain (RQC). Using multi-dimensional NMR spectroscopy, we have determined the solution structure of BLM RQC, and found that BLM RQC retains the overall winged-helix motif previously observed for other RQC proteins. Comparison between BLM RQC and the RQC domain of its homologue, Werner syndrome protein (WRN RQC), revealed two major structural differences. Firstly, BLM RQC contains an extended 14-residue insertion forming a flexible loop between two first α-helices, only found in BLM RQC and not other RQC proteins. Secondly, in contrast to the third α-helix of WRN RQC, an unstructured loop was observed for this region of BLM RQC.     

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.     

Structure and mechanism of error-free replication past the major benzo[a]pyrene adduct by human DNA polymerase κ

Benzo[a]pyrene (BP) is a well-known and frequently encountered carcinogen which generates a bulky DNA adduct (+)-trans-10S-BP-N²-dG (BP-dG) in cells. DNA polymerase kappa (polκ) is the only known Y-family polymerase that bypasses BP-dG accurately and thus protects cells from genotoxic BP. Here, we report the structures of human polκ in complex with DNA containing either a normal guanine (G) base or a BP-dG adduct at the active site and a correct deoxycytidine. The structures and supporting biochemical data reveal a unique mechanism for accurate replication by translesion synthesis past the major bulky adduct. The active site of polκ opens at the minor groove side of the DNA substrate to accommodate the bulky BP-dG that is attached there. More importantly, polκ stabilizes the lesion DNA substrate in the same active conformation as for regular B-form DNA substrates and the bulky BPDE ring in a 5′ end pointing conformation. The BP-dG adducted DNA substrate maintains a Watson–Crick (BP-dG:dC) base pair within the active site, governing correct nucleotide insertion opposite the bulky adduct. In addition, polκ''s unique N-clasp domain supports the open conformation of the enzyme and the extended conformation of the single-stranded template to allow bypass of the bulky lesion. This work illustrates the first molecular mechanism for how a bulky major adduct is replicated accurately without strand misalignment and mis-insertion.     

Translesion replication of benzo[a]pyrene and benzo[c]phenanthrene diol epoxide adducts of deoxyadenosine and deoxyguanosine by human DNA polymerase iota

Human DNA polymerase ι (polι) is a Y-family polymerase whose cellular function is presently unknown. Here, we report on the ability of polι to bypass various stereoisomers of benzo[a]pyrene (BaP) diol epoxide (DE) and benzo[c]phenanthrene (BcPh) DE adducts at deoxyadenosine (dA) or deoxyguanosine (dG) bases in four different template sequence contexts in vitro. We find that the BaP DE dG adducts pose a strong block to polι-dependent replication and result in a high frequency of base misincorporations. In contrast, misincorporations opposite BaP DE and BcPh DE dA adducts generally occurred with a frequency ranging between 2 × 10^–3 and 6 × 10^–4. Although dTMP was inserted efficiently opposite all dA adducts, further extension was relatively poor, with one exception (a cis opened adduct derived from BcPh DE) where up to 58% extension past the lesion was observed. Interestingly, another human Y-family polymerase, polκ, was able to extend dTMP inserted opposite a BaP DE dA adduct. We suggest that polι might therefore participate in the error-free bypass of DE-adducted dA in vivo by predominantly incorporating dTMP opposite the damaged base. In many cases, elongation would, however, require the participation of another polymerase more specialized in extension, such as polκ.     

Role of AtPol ζ, AtRev1 and AtPolη in γ ray-induced mutagenesis

Although ionizing radiation has been employed as a mutagenic agent in plants, the molecular mechanism(s) of the mutagenesis is poorly understood. AtPolζ, AtRev1 and AtPolη are Arabidopsis translesion synthesis (TLS)-type polymerases involved in UV-induced mutagenesis. To investigate the role of TLS-type DNA polymerases in radiation-induced mutagenesis, we analyzed the mutation frequency in AtPolζ-, AtRev1- or AtPolη-knockout plants rev3-1, rev1-1 and polh-1, respectively. The change in mutation frequency in rev3-1 was negligible, whereas that in rev1-1 decreased markedly and that in polh-1 increased slightly compared to wild-type. Abasic (apurinic/apyrimidinic; AP) sites, induced by radiation or generated during DNA repair processes, can pair with any kind of nucleotide on the opposite strand. 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dG), induced by radiation following formation of reactive oxygen species, can pair with cytosine or adenine. Therefore, AtRev1 possibly inserts dC opposite an AP site or 8-oxo-dG, which results in G to T transversions.Key words: ionizing radiation, DNA damage, translesion synthesis, ROS, 8-oxo-dG, ArabidopsisIonizing radiation has been applied to various plants for the purpose of generating useful agricultural resources. A variety of ionizing radiation forms, including X rays, γ rays, neutrons and ion-beams, have been used as mutagens for mutation breeding in addition to chemical mutagens.¹ Nevertheless, the molecular mechanism(s) associated with radiation-induced mutations in higher plants remains to be fully understood.In animals and microorganisms, it is known that a large proportion of mutations occur when damaged DNA is replicated by specific DNA polymerases. This activity is referred to as “translesion synthesis (TLS),” and represents one of the damage-tolerance pathways conserved from bacteria to humans. TLS-type polymerases have a more relaxed active site structure compared to replicases and therefore can act on damaged templates. However, the very flexible nature of the active site can induce high and sometimes fatal, replication errors. In higher plants, the presence of several TLS-type polymerase genes was reported. AtREV3 encodes the catalytic subunit of AtPolζ.² AtPOLK, AtREV1 and AtPOLH encode AtPolκ, AtRev1 and AtPolη, respectively.^3–7 In our previous paper, we suggested the role of three TLS-type polymerases, AtPolζ, AtRev1 and AtPolη, in the formation of UV-induced mutations.⁸Since the variety and ratio of UV-induced DNA damage have been well characterized, and the TLS activity of each polymerase can be examined in vitro, it is relatively easy to speculate on how the TLS polymerases induce mutation following UV-exposure. By contrast, ionizing radiation can induce a variety of damage, including damage to bases and strand breaks, and the role of TLS-type polymerases in radiation-induced mutation is less understood.In an effort to determine whether TLS polymerases are involved in radiation-induced mutation in higher plants, we analyzed the mutation frequency in Arabidopsis somatic tissues following γ ray irradiation. The reporter gene used for this analysis was the uidA_166G-T gene, which contains a nonsense mutation generated by replacement of the 166^th guanine with thymine.⁹ The reporter gene integrated in the Arabidopsis genome will become active when a T-to-G reversion occurs at the 166^ththymine. To detect γ ray-induced mutations, transgenic plants carrying the uidA_166G-T were treated with 100 Gy of γ rays and then grown for another 10 days, so that cells with an active uidA gene can proliferate and produce a detectable blue sector on somatic tissues.To investigate the roles of TLS-type polymerases in radiation-induced mutations, we examined the mutation frequencies in disruptants of the AtREV3, AtREV1 and AtPOLH genes, rev3-1, rev1-1 and polh-1, respectively, and compared these to that of wild-type. The reversion events in rev3-1 did not change significantly compared to wild-type siblings (Fig. 1). This is contrasted with the reduction in UV-induced mutation frequency when AtPolζ is disrupted.⁸ However, the reversion events in rev1-1 plants were less than 1/10 of that in wild-type siblings (p < 0.01). This result indicates that AtRev1 plays a role in promoting γ ray-induced mutations. The reversion event in polh-1 was slightly (∼1.4 times) higher than that in wild-type siblings (p < 0.05), suggesting that AtPolη plays a role in reducing γ ray-induced mutations.Open in a separate windowFigure 1γ ray-induced mutation frequencies in AtREV3-, AtREV1- and AtPOLH-disrupted plants. Wild-type and mutant derived from a single F1 plant were examined concurrently. Bars represent average frequencies per 100 plants derived from multiple experiments. error bars indicate ±SE. *p < 0.01; **p < 0.05.The frequencies in wild-type, rev3-1, rev1-1 and polh-1 were 12, 22, 1.9 and 13 times higher, respectively, with γ ray exposure compared to the spontaneous mutation frequency as previously reported.⁸ These results indicate that the G to T transversion was greatly induced by γ ray exposure.Since ionizing radiation can induce a variety of damage to DNA or nucleotide pools, the mechanisms associated with radiation-induced mutagenesis would be more complicated than those pertaining to UV-induced mutagenesis. It is known that some kinds of damage are more abundantly generated by ionizing radiation. Additionally, some kinds of damage are preferentially used as templates or substrates by specific DNA polymerases. Based on previous reports relating to plants or other organisms, we propose two possible mechanisms to account for the γ ray-induced reversion events (Fig. 2).Open in a separate windowFigure 2Possible role of TLS polymerases in γ ray-induced mutagenesis. (A) role of TLS polymerases in the replication of AP sites. Ionizing radiation induces formation of an AP site (O). AtRev1 inserts dC opposite the AP site, leading to a G to T transversion. AtPolη inserts dA or T opposite the AP site, contributing less to G to T transversions. (B) Ionizing radiation induces the formation of reactive oxygen species (ROS) which oxidize guanine (G) in DNA or dGTP, producing 8-oxo-dG or 8-oxo-dGTP (G^o). 8-oxo-dGTP is misincorporated opposite adenine (A) through replication. G^o is paired with cytosine (C) at the next round of DNA replication, which results in a T to G transversion. AtPolη inserts dC or dA opposite G^o, whereas AtRev1 inserts dC opposite G^o. Other polymerases including AtPolκ might insert dA opposite G^o.Abasic (apurinic/apyrimidinic; AP) sites represent one of the most abundant DNA lesions that occur spontaneously and are induced by radiation.¹⁰ AP sites can also be generated during the DNA repair process.¹¹ If the 166^th T of our marker gene were lost following irradiation with γ rays, the template would induce various mutations.Among the TLS-type polymerases, Rev1s share the specific ability to insert dCMP opposite AP sites.^12–14 Therefore, the significant reduction in mutation frequency in AtRev1-knockout plants might be due to loss of dCMP insertion opposite AP sites (Fig. 2A). In contrast, it was shown that yeast or human Polηs insert dA or T opposite AP sites or AP-site analogs.^15–17 Thus, the activity of Polη does not seem to contribute toward T to G transversions (Fig. 2A). The incidence of mutagenic bypass of AP sites by AtRev1 may be greater when AtPolη is absent, which elevates the mutation frequency slightly.Given the similar reduction in UV-induced mutation frequencies, we previously suggested that AtRev1 cooperates with AtPolζ to bypass UV-damage.⁸ In contrast, no significant change in γ ray-induced mutation frequency was observed in AtPolζ-knockout plants. This suggests that AtRev1 might work independently of AtPolζ when bypassing AP sites, although it is not consistent with previous reports concerning yeast.^15,16Radiation damages cells through the formation of reactive oxygen species (ROS). ROS induce oxidative damage of DNA, including strand breaks and base and nucleotide modifications. The formation of 7,8-dihydro-8-oxo-2′-deoxy-guanosine (8-oxo-dG) represents one of the most abundant and best characterized type of oxidative damage.¹⁸ 8-oxo-dG can pair with cytosine or adenine, inducing frequent base substitutions. In addition to direct oxidation of deoxyguanosine (dG) in DNA, 8-oxo-dG can be generated by the incorporation of oxidized dGTP (8-oxo-dGTP) into DNA during the replication process.¹⁹ 8-oxo-dG in DNA induces mutations when used as a template for the next round of replication. If 8-oxo-dGTP were incorporated in lieu of the 166^thT and paired with dC in the next round of replication, it would lead to a T to G transversion (Fig. 2B).It was shown that yeast and human Rev1s insert dC at positions opposite 8-oxo-dG.^13,20 Therefore, the reduction in mutation frequency in AtRev1-knockout plants could be due to loss of dCMP insertion opposite 8-oxo-dG (Fig. 2B). Although human and yeast Polηs can insert dC or dA opposite 8-oxo-dG, the insertion efficiencies and dC/dA ratios differ depending on the assay conditions and sequence context.^21–25 Thus, the balance of error-free and error-prone bypass activities of Polη might interfere with the mutation frequency in individual assays. The slight increase in mutation frequency in AtPolη-knockout plants suggests that the ratio of dC insertion by other polymerases was slightly higher when AtPolη is absent.In yeast, spontaneous mutations in base excision repair (BER)-deficient cells are not reduced by elimination of Polζ, suggesting a minor role of Polζ in 8-oxo-dG induced mutations.^26,27 Our result demonstrating no reduction in mutation frequency in AtPolζ-knockout plants suggests that AtPolζ is also dispensable in terms of 8-oxo-dG induced mutagenesis. However, the root growth of AtPolζ-knockout plants is severely inhibited by γ ray exposure.^2,4 Therefore, it is possible that AtPolζ has other function(s) in radiation-induced damage responses.In addition to the three polymerases examined in this report, Arabidopsis possesses an additional TLS-type polymerase referred to as AtPolκ. In vitro analysis revealed that AtPolκ preferentially inserts dA opposite 8-oxo-dG,²⁸ as is the case with human Polκ.^29,30 Therefore, it is conceivable that AtPolκ has a function to promote T to G transversions (Fig. 2B). It will be interesting to measure the mutation frequency in AtPolκ-knockout plants following γ ray exposure. Further, analyses of mutation frequencies in BER- or mismatch repair (MMR)-deficient mutants will be necessary to delineate the mechanism(s) of radiation-induced mutagenesis in higher plants.     

The human Werner syndrome protein stimulates repair of oxidative DNA base damage by the DNA glycosylase NEIL1

The mammalian DNA glycosylase, NEIL1, specific for repair of oxidatively damaged bases in the genome via the base excision repair pathway, is activated by reactive oxygen species and prevents toxicity due to radiation. We show here that the Werner syndrome protein (WRN), a member of the RecQ family of DNA helicases, associates with NEIL1 in the early damage-sensing step of base excision repair. WRN stimulates NEIL1 in excision of oxidative lesions from bubble DNA substrates. The binary interaction between NEIL1 and WRN (K(D) = 60 nM) involves C-terminal residues 288-349 of NEIL1 and the RecQ C-terminal (RQC) region of WRN, and is independent of the helicase activity WRN. Exposure to oxidative stress enhances the NEIL-WRN association concomitant with their strong nuclear co-localization. WRN-depleted cells accumulate some prototypical oxidized bases (e.g. 8-oxoguanine, FapyG, and FapyA) indicating a physiological function of WRN in oxidative damage repair in mammalian genomes. Interestingly, WRN deficiency does not have an additive effect on in vivo damage accumulation in NEIL1 knockdown cells suggesting that WRN participates in the same repair pathway as NEIL1.     

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.     

Solution structure of the RecQ C-terminal domain of human Bloom syndrome protein

RecQ C-terminal (RQC) domain is known as the main DNA binding module of RecQ helicases such as Bloom syndrome protein (BLM) and Werner syndrome protein (WRN) that recognizes various DNA structures. Even though BLM is able to resolve various DNA structures similarly to WRN, BLM has different binding preferences for DNA substrates from WRN. In this study, we determined the solution structure of the RQC domain of human BLM. The structure shares the common winged-helix motif with other RQC domains. However, half of the N-terminal has unstructured regions (α1–α2 loop and α3 region), and the aromatic side chain on the top of the β-hairpin, which is important for DNA duplex strand separation in other RQC domains, is substituted with a negatively charged residue (D1165) followed by the polar residue (Q1166). The structurally distinctive features of the RQC domain of human BLM suggest that the DNA binding modes of the BLM RQC domain may be different from those of other RQC domains.     

Structure of Human DNA Polymerase κ Inserting dATP Opposite an 8-OxoG DNA Lesion

Background

Oxygen-free radicals formed during normal aerobic cellular metabolism attack bases in DNA and 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the major lesions formed. It is amongst the most mutagenic lesions in cells because of its dual coding potential, wherein 8-oxoG(syn) can pair with an A in addition to normal base pairing of 8-oxoG(anti) with a C. Human DNA polymerase κ (Polκ) is a member of the newly discovered Y-family of DNA polymerases that possess the ability to replicate through DNA lesions. To understand the basis of Polκ''s preference for insertion of an A opposite 8-oxoG lesion, we have solved the structure of Polκ in ternary complex with a template-primer presenting 8-oxoG in the active site and with dATP as the incoming nucleotide.

Methodology and Principal Findings

We show that the Polκ active site is well-adapted to accommodate 8-oxoG in the syn conformation. That is, the polymerase and the bound template-primer are almost identical in their conformations to that in the ternary complex with undamaged DNA. There is no steric hindrance to accommodating 8-oxoG in the syn conformation for Hoogsteen base-paring with incoming dATP.

Conclusions and Significance

The structure we present here is the first for a eukaryotic translesion synthesis (TLS) DNA polymerase with an 8-oxoG:A base pair in the active site. The structure shows why Polκ is more efficient at inserting an A opposite the 8-oxoG lesion than a C. The structure also provides a basis for why Polκ is more efficient at inserting an A opposite the lesion than other Y-family DNA polymerases.     

Mutational specificity of gamma-radiation-induced guanine-thymine and thymine-guanine intrastrand cross-links in mammalian cells and translesion synthesis past the guanine-thymine lesion by human DNA polymerase eta

Comparative mutagenesis of gamma- or X-ray-induced tandem DNA lesions G[8,5-Me]T and T[5-Me,8]G intrastrand cross-links was investigated in simian (COS-7) and human embryonic (293T) kidney cells. For G[8,5-Me]T in 293T cells, 5.8% of progeny contained targeted base substitutions, whereas 10.0% showed semitargeted single-base substitutions. Of the targeted mutations, the G --> T mutation occurred with the highest frequency. The semitargeted mutations were detected up to two bases 5' and three bases 3' to the cross-link. The most prevalent semitargeted mutation was a C --> T transition immediately 5' to the G[8,5-Me]T cross-link. Frameshifts (4.6%) (mostly small deletions) and multiple-base substitutions (2.7%) also were detected. For the T[5-Me,8]G cross-link, a similar pattern of mutations was noted, but the mutational frequency was significantly higher than that of G[8,5-Me]T. Both targeted and semitargeted mutations occurred with a frequency of approximately 16%, and both included a dominant G --> T transversion. As in 293T cells, more than twice as many targeted mutations in COS cells occurred in T[5-Me,8]G (11.4%) as in G[8,5-Me]T (4.7%). Also, the level of semitargeted single-base substitutions 5' to the lesion was increased and 3' to the lesion decreased in T[5-Me,8]G relative to G[8,5-Me]T in COS cells. It appeared that the majority of the base substitutions at or near the cross-links resulted from incorporation of dAMP opposite the template base, in agreement with the so-called "A-rule". To determine if human polymerase eta (hpol eta) might be involved in the mutagenic bypass, an in vitro bypass study of G[8,5-Me]T in the same sequence was carried out, which showed that hpol eta can bypass the cross-link incorporating the correct dNMP opposite each cross-linked base. For G[8,5-Me]T, nucleotide incorporation by hpol eta was significantly different from that by yeast pol eta in that the latter was more error-prone opposite the cross-linked Gua. The incorporation of the correct nucleotide, dAMP, by hpol eta opposite cross-linked T was 3-5-fold more efficient than that of a wrong nucleotide, whereas incorporation of dCMP opposite the cross-linked G was 10-fold more efficient than that with dTMP. Therefore, the nucleotide incorporation pattern by hpol eta was not consistent with the observed cellular mutations. Nevertheless, at and near the lesion, hpol eta was more error-prone compared to a control template. The in vitro data suggest that translesion synthesis by another Y-family DNA polymerase and/or flawed participation of an accessory protein is a more likely scenario in the mutagenesis of these lesions in mammalian cells. However, hpol eta may play a role in correct bypass of the cross-links.