Bypass Of Adducts Research Articles

Strand-specific PCR-competitive replication and adduct bypass assay for assessing how DNA adducts perturb DNA replication in mammalian cells.

Human genomes are susceptible to damage by a variety of endogenous and exogenous agents. If not repaired, the resulting DNA lesions can potentially lead to mutations, genome instability, and cell death. While existing in vitro experiments allow for characterizing replication outcomes from the use of purified translesion synthesis (TLS) DNA polymerases, such studies often lack the sophistication and dynamic nature of cellular contexts. Here, we present a strand-specific PCR-based Competitive Replication and Adduct Bypass (ssPCR-CRAB) assay designed to investigate quantitatively the impact of DNA lesions on replication efficiency and fidelity in mammalian cells. Combined with genetic manipulation, this approach facilitates the revelation of diverse functions of TLS polymerases in replication across DNA lesions.

DNA Polymerase η Promotes the Transcriptional Bypass of N2-Alkyl-2'-deoxyguanosine Adducts in Human Cells.

To cope with unrepaired DNA lesions, cells are equipped with DNA damage tolerance mechanisms, including translesion synthesis (TLS). While TLS polymerases are well documented in facilitating replication across damaged DNA templates, it remains unknown whether TLS polymerases participate in transcriptional bypass of DNA lesions in cells. Herein, we employed the competitive transcription and adduct bypass assay to examine the efficiencies and fidelities of transcription across N2-alkyl-2'-deoxyguanosine (N2-alkyl-dG, alkyl = methyl, ethyl, n-propyl, or n-butyl) lesions in HEK293T cells. We found that N2-alkyl-dG lesions strongly blocked transcription and elicited CC → AA tandem mutations in nascent transcripts, where adenosines were misincorporated opposite the lesions and their adjacent 5' nucleoside. Additionally, genetic ablation of Pol η, but not Pol κ, Pol ι, or Pol ζ, conferred marked diminutions in the transcriptional bypass efficiencies of the N2-alkyl-dG lesions, which is exacerbated by codepletion of Rev1 in Pol η-deficient background. We also observed that the repair of N2-nBu-dG was not pronouncedly affected by genetic depletion of Pol η or Rev1. Hence, our results provided insights into transcriptional perturbations induced by N2-alkyl-dG lesions and expanded the biological functions of TLS DNA polymerases.

Repair and translesion synthesis of O6-alkylguanine DNA lesions in human cells

O6-alkyl-2'-deoxyguanosine (O6-alkyl-dG) lesions are among the most mutagenic and prevalent alkylated DNA lesions that are associated with cancer initiation and progression. In this study, using a shuttle vector-based strand-specific PCR-competitive replication and adduct bypass assay in conjunction with tandem MS for product identification, we systematically assessed the repair and replicative bypass of a series of O6-alkyl-dG lesions, with the alkyl group being a Me, Et, nPr, iPr, nBu, iBu, or sBu, in several human cell lines. We found that the extent of replication-blocking effects of these lesions is influenced by the size of the alkyl groups situated on the O6 position of the guanine base. We also noted involvement of distinct DNA repair pathways and translesion synthesis polymerases (Pols) in ameliorating the replication blockage effects elicited by the straight- and branched-chain O6-alkyl-dG lesions. We observed that O6-methylguanine DNA methyltransferase is effective in removing the smaller alkyl groups from the O6 position of guanine, whereas repair of the branched-chain lesions relied on nucleotide excision repair. Moreover, these lesions were highly mutagenic during cellular replication and exclusively directed G→A mutations; Pol η and Pol ζ participated in error-prone bypass of the straight-chain lesions, whereas Pol κ preferentially incorporated the correct dCMP opposite the branched-chain lesions. Together, these results uncover key cellular proteins involved in repair and translesion synthesis of O6-alkyl-dG lesions and provide a better understanding of the roles of these types of lesions in the etiology of human cancer.

DNA Adduct-Directed Synthetic Nucleosides.

Chemical damage to DNA is a key initiator of adverse biological consequences due to disruption of the faithful reading of the genetic code. For example, O6-alkylguanine ( O6-alkylG) DNA adducts are strongly miscoding during DNA replication when the damaged nucleobase is a template for polymerase-mediated translesion DNA synthesis. Thus, mutations derived from O6-alkylG adducts can have severe adverse effects on protein translation and function and are an early event in the initiation of carcinogenesis. However, the low abundance of these adducts places significant limitations on our ability to relate their presence and biological influences with resultant mutations or disease risk. As a consequence, there is a critical need for novel tools to detect and study the biological role of alkylation adducts. Incorporating DNA bases with altered structures that are derived synthetically is a strategy that has been used widely to interrogate biological processes involving DNA. Such synthetic nucleosides have contributed to our understanding of DNA structure, DNA polymerase (Pol) and repair enzyme function, and to the expansion of the genetic alphabet. This Account describes our efforts toward creating and applying synthetic nucleosides directed at DNA adducts. We synthesized a variety of nucleosides with altered base structures that complement the altered hydrogen bonding capacity and hydrophilicity of O6-alkylG adducts. The heterocyclic perimidinone-derived nucleoside Per was the first of such adduct-directed synthetic nucleosides; it specifically stabilized O6-benzylguanine ( O6-BnG) in a DNA duplex. Structural variants of Per were used to determine hydrogen bonding and base-stacking contributions to DNA duplex stability in templates containing O6-BnG as well as O6-methylguanine ( O6-MeG) adducts. We created synthetic probes able to stabilize damaged over undamaged templates and established how altered hydrogen bonding or base-stacking properties impact DNA duplex stability as a function of adduct structures. This knowledge was then applied to devise a hybridization-based detection strategy involving gold nanoparticles that distinguish damaged from undamaged DNA by colorimetric changes. Furthermore, synthetic nucleosides were used as mechanistic tools to understand chemical determinants such as hydrogen bonding, π-stacking, and size and shape deviations that impact the efficiency and fidelity of DNA adduct bypass by DNA Pols. Finally, we reported the first example of amplifying alkylated DNA, accomplished by combining an engineered polymerase and synthetic triphosphate for which incorporation is templated by a DNA adduct. The presence of the synthetic nucleoside in amplicons could serve as a marker for the presence and location of DNA damage at low levels in DNA strands. Adduct-directed synthetic nucleosides have opened new concepts to interrogate the levels, locations, and biological influences of DNA alkylation.

DNA replication studies of N-nitroso compound–induced O6-alkyl-2′-deoxyguanosine lesions in Escherichia coli

N-Nitroso compounds (NOCs) are common DNA-alkylating agents, are abundantly present in food and tobacco, and can also be generated endogenously. Metabolic activation of some NOCs can give rise to carboxymethylation and pyridyloxobutylation/pyridylhydroxybutylation of DNA, which are known to be carcinogenic and can lead to gastrointestinal and lung cancer, respectively. Herein, using the competitive replication and adduct bypass (CRAB) assay, along with MS- and NMR-based approaches, we assessed the cytotoxic and mutagenic properties of three O6-alkyl-2'-deoxyguanosine (O6-alkyl-dG) adducts, i.e. O6-pyridyloxobutyl-dG (O6-POB-dG) and O6-pyridylhydroxybutyl-dG (O6-PHB-dG), derived from tobacco-specific nitrosamines, and O6-carboxymethyl-dG (O6-CM-dG), induced by endogenous N-nitroso compounds. We also investigated two neutral analogs of O6-CM-dG, i.e. O6-aminocarbonylmethyl-dG (O6-ACM-dG) and O6-hydroxyethyl-dG (O6-HOEt-dG). We found that, in Escherichia coli cells, these lesions mildly (O6-POB-dG), moderately (O6-PHB-dG), or strongly (O6-CM-dG, O6-ACM-dG, and O6-HOEt-dG) impede DNA replication. The strong blockage effects of the last three lesions were attributable to the presence of hydrogen-bonding donor(s) located on the alkyl functionality of these lesions. Except for O6-POB-dG, which also induced a low frequency of G → T transversions, all other lesions exclusively stimulated G → A transitions. SOS-induced DNA polymerases played redundant roles in bypassing all the O6-alkyl-dG lesions investigated. DNA polymerase IV (Pol IV) and Pol V, however, were uniquely required for inducing the G → A transition for O6-CM-dG exposure. Together, our study expands our knowledge about the recognition of important NOC-derived O6-alkyl-dG lesions by the E. coli DNA replication machinery.

TLS dependent and independent functions of DNA polymerase eta (Polη/Rad30) from Pathogenic Yeast Candida albicans

Polη, a unique TLS DNA polymerase that promotes efficient bypass of UV-induced CPDs and cisplatin adducts, has not been explored in Candida species yet. Here, we show that CaPolη plays a vital role in protecting Candida albicans genome from diverse array of DNA damaging agents, not limited to UV and cisplatin. Polη deficient strain did not exhibit any hyphal development in the presence of UV and cisplatin while the wild type strain profusely developed DNA damage induced filamentation. The polarized growth induced by HU and MMS was found to be Polη independent. No common regulatory pathway of morphogenesis operates in C. albicans due to genomic stress, rather Polη branches away from RAD53 dependent pathway to be specific to UV/cisplatin. Interestingly, serum that does not inflict any DNA damage also induces hyphal growth in C. albicans, and requires a functionally active Polη. Importantly, deletion of RAD30 sensitized the strain to amphotericin B; but its presence resulted in azole drug tolerance only in DNA damaging conditions. We suggest that the roles of CaPolη in genome stability and genotoxins induced filamentation are due to its TLS activities; whereas its TLS independent functions play a vital role in serum induced morphogenesis and amphotericin B resistance.

Structural basis for proficient nucleotide incorporation across a major cisplatin‐DNA lesion by human DNA polymerase kappa (polκ)

Cisplatin (cis‐diamminedichloroplatinum) is a widely used chemotherapeutic compound. It reacts with the N7 atoms of adjacent guanines in DNA to form the Pt‐1,2‐d(GpG) intrastrand cross‐link (Pt‐GG) as a major product. This bulky cisplatin adduct blocks DNA replication by replicative polymerases, thereby promoting apoptosis of rapidly dividing cancer cells. However, cancer cells have developed resistance to cisplatin, partly because of the bypass of cisplatin adducts by specialized DNA polymerases (mostly belonging to the Y‐family) through translesion synthesis (TLS). The human Y‐family DNA polymerase eta (polη) is very efficient in replicating opposite the Pt‐GG lesions, but not in extending after this adduct. Recently, it was shown that human Y‐family DNA polymerase kappa (polκ) is indispensable for DNA repair synthesis in dorsal root ganglion neurons exposed to cisplatin. Polκ gene knockdown experiments in human cells have also indicated the involvement of polκ in TLS of the Pt‐GG lesion. In this study, we investigated the bypass potential and nucleotide incorporation preference of polκ opposite DNA containing the Pt‐GG adduct and determined two crystal structures of polκ complexed with such DNA. The ternary complex structures represent two consecutive stages of lesion bypass: nucleotide insertion opposite the 5′G (Pt‐GG2) and primer extension immediately after the lesion (Pt‐GG3). Our in vitro DNA replication assays using polκ and DNA containing the Pt‐GG lesion indicate that polκ inserts the correct dCTP opposite the 5′G of Pt‐GG (Pt‐GG2), when a nucleotide is already inserted opposite the 3′G (Pt‐GG1). Polκ also inserts the correct dATP opposite the T base immediately after the Pt‐GG (Pt‐GG3). However, polκ is not able to insert a nucleotide opposite the 3′G of Pt‐GG (Pt‐GG1). On the other hand, polη is very efficient in replicating the Pt‐GG lesion at two insertion stages (Pt‐GG1 and Pt‐GG2), but the enzyme is blocked at the extension stage (Pt‐GG3). In terms of fidelity, we found that polκ has the fewest mis‐insertions compared to two other human Y‐family polymerases polη and polι. Our steady‐state kinetic analysis showed that polκ incorporates dCTP and dATP opposite the 5′G of Pt‐GG (Pt‐GG2) and T template 5′ to the lesion (Pt‐GG3), respectively, with very similar relative efficiencies of 0.15–0.20, which are only ~5 fold lower than for normal DNA replication (GG2 and GG3). Our biochemical data indicate that polκ is very proficient and accurate in inserting a nucleotide after the first G of the Pt‐GG lesion. The structures revealed that the accuracy is achieved by stably accommodating the bases containing the cisplatin adducts in the enzyme's active site for proper Watson‐Crick base pairing with the incoming nucleotide. Together, our biochemical and structural data provide the molecular basis for the accurate and proficient incorporation by polκ of nucleotides opposite the cisplatin adduct and suggest an important role of polκ as a second polymerase (extender) in the efficient bypass of the Pt‐GG lesion in vivo. This work holds promise for polκ as a potential target for drug design, along with polη, which together could improve the efficacy of cisplatin treatment for cancer therapy.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cytotoxic and Mutagenic Properties of C3'-Epimeric Lesions of 2'-Deoxyribonucleosides in Escherichia coli Cells.

Reactive oxygen species (ROS), resulting from endogenous metabolism and/or environmental exposure, can induce damage to the 2-deoxyribose moiety in DNA. Specifically, a hydrogen atom from each of the five carbon atoms in 2-deoxyribose can be abstracted by hydroxyl radical, and improper chemical repair of the ensuing radicals formed at the C1', C3', and C4' positions can lead to the stereochemical inversion at these sites to yield epimeric 2-deoxyribose lesions. Although ROS-induced single-nucleobase lesions have been well studied, the biological consequences of the C3'-epimeric lesions of 2'-deoxynucleosides, i.e., 2'-deoxyxylonucleosides (dxN), have not been comprehensively investigated. Herein, we assessed the impact of dxN lesions on the efficiency and fidelity of DNA replication in Escherichia coli cells by conducting a competitive replication and adduct bypass assay with single-stranded M13 phage containing a site-specifically incorporated dxN. Our results revealed that, of the four dxN lesions, only dxG constituted a strong impediment to DNA replication, and intriguingly, dxT and dxC conferred replication bypass efficiencies higher than those of the unmodified counterparts. In addition, the three SOS-induced DNA polymerases (Pol II, Pol IV, and Pol V) did not play any appreciable role in bypassing these lesions. Among the four dxNs, only dxA directed a moderate frequency of dCMP misincorporation. These results provided important insights into the impact of the C3'-epimeric lesions on DNA replication in E. coli cells.

Minor Groove 3-Deaza-Adenosine Analogues: Synthesis and Bypass in Translesion DNA Synthesis.

Anticancer drugs that alkylate DNA in the minor groove may give rise to 3-alkyl-adenosine adducts that interfere with replication, inducing apoptosis in rapidly dividing cancer cells. However, translesion DNA synthesis (TLS) by polymerase enzymes (Pols) with the capacity to bypass DNA adducts may contribute to damage tolerance and drug resistance. 3-Alkyl-adenosine adducts are unstable and depurinate, which is a barrier to addressing chemical and enzymatic aspects of how they impact the progress of DNA Pols. To characterize structure-based relationships of 3-adenine alkylation relevant to cancer drugs on duplex stability and DNA Pol-catalyzed DNA synthesis, we synthesized stable 3-deaza-3-alkyl-adenosine analogues, including 3-deaza-3-phenethyl-adenosine and 3-deaza-3-methoxynaphthylethyl-adenosine, and incorporated them into oligonucleotides. A moderate reduction of duplex stability was observed on the basis of thermal denaturation data. Replication studies using purified Y-family human DNA Pols hPol η, κ, and ι indicated that these enzymes can perform TLS over the modified bases. hPol η had higher misincorporation rates when synthesizing opposite the modified bases compared with adenine, whereas hPol κ and ι maintained high fidelity. These results provide insight into how alterations in chemical structure reduce bypass of minor-groove adducts, and provide novel chemical probes for evaluating minor-groove DNA alkylation.

Specific incorporation of an artificial nucleotide opposite a mutagenic DNA adduct by a DNA polymerase.

The ability to detect DNA modification sites at single base resolution could significantly advance studies regarding DNA adduct levels, which are extremely difficult to determine. Artificial nucleotides that are specifically incorporated opposite a modified DNA site offer a potential strategy for detection of such sites by DNA polymerase-based systems. Here we investigate the action of newly synthesized base-modified benzimidazole-derived 2'-deoxynucleoside-5'-O-triphosphates on DNA polymerases when performing translesion DNA synthesis past the pro-mutagenic DNA adduct O(6)-benzylguanine (O(6)-BnG). We found that a mutated form of KlenTaq DNA polymerase, i.e., KTqM747K, catalyzed O(6)-BnG adduct-specific processing of the artificial BenziTP in favor of the natural dNTPs. Steady-state kinetic parameters revealed that KTqM747K catalysis of BenziTP is 25-fold more efficient for template O(6)-BnG than G, and 5-fold more efficient than natural dTMP misincorporation in adduct bypass. Furthermore, the nucleotide analogue BenziTP is required for full-length product formation in O(6)-BnG bypass, as without BenziTP the polymerase stalls at the adduct site. By combining the KTqM747K polymerase and BenziTP, a first round of DNA synthesis enabled subsequent amplification of Benzi-containing DNA. These results advance the development of technologies for detecting DNA adducts.

Effects of Tet-mediated oxidation products of 5-methylcytosine on DNA transcription in vitro and in mammalian cells.

5-methylcytosine (5-mC) is a well-characterized epigenetic regulator in mammals. Recent studies showed that Ten-eleven translocation (Tet) proteins can catalyze the stepwise oxidation of 5-mC to produce 5-hydroxymethylcytosine (5-HmC), 5-formylcytosine (5-FoC) and 5-carboxylcytosine (5-CaC). The exciting discovery of these novel cytosine modifications has stimulated substantial research interests about their roles in epigenetic regulation. Here we systematically examined the effects of the oxidized 5-mC derivatives on the efficiency and fidelity of DNA transcription using a recently developed competitive transcription and adduct bypass assay. Our results showed that, when located on the transcribed strand, 5-FoC and 5-CaC exhibited marginal mutagenic and modest inhibitory effects on DNA transcription mediated by single-subunit T7 RNA polymerase or multi-subunit human RNA polymerase II in vitro and in human cells. 5-HmC displayed relatively milder blocking effects on transcription, and no mutant transcript could be detectable for 5-HmC in vitro or in cells. The lack of considerable mutagenic effects of the oxidized 5-mC derivatives on transcription was in agreement with their functions in epigenetic regulation. The modest blocking effects on transcription suggested that 5-FoC and 5-CaC may function in transcriptional regulation. These findings provided new evidence for the potential functional interplay between cytosine methylation status and transcription.

Effects of 6-Thioguanine and S6-Methylthioguanine on Transcription in Vitro and in Human Cells

Thiopurine drugs are extensively used as chemotherapeutic agents in clinical practice, even though there is concern about the risk of therapy-related cancers. It has been previously suggested that the cytotoxicity of thiopurine drugs involves their metabolic activation, the resultant generation of 6-thioguanine ((S)G) and S(6)-methylthioguanine (S(6)mG) in DNA, and the futile mismatch repair triggered by replication-induced (S)G:T and S(6)mG:T mispairs. Disruption of transcription is known to be one of the major consequences of DNA damage induced by many antiviral and antitumor agents; however, it remains undefined how (S)G and S(6)mG compromise the efficiency and fidelity of transcription. Using our recently developed competitive transcription and adduct bypass assay, herein we examined the impact of (S)G and S(6)mG on transcription in vitro and in human cells. Our results revealed that, when situated on the transcribed strand, S(6)mG exhibited both inhibitory and mutagenic effects during transcription mediated by single-subunit T7 RNA polymerase or multisubunit human RNA polymerase II in vitro and in human cells. Moreover, we found that the impact of S(6)mG on transcriptional efficiency and fidelity is modulated by the transcription-coupled nucleotide excision repair capacity. In contrast, (S)G did not considerably compromise the efficiency or fidelity of transcription, and it was a poor substrate for NER. We propose that S(6)mG might contribute, at least in part, to thiopurine-mediated cytotoxicity through inhibition of transcription and to potential therapy-related carcinogenesis via transcriptional mutagenesis.

A quantitative assay for assessing the effects of DNA lesions on transcription

Most mammalian cells in nature are quiescent but actively transcribing mRNA for normal physiological processes; thus, it is important to investigate how endogenous and exogenous DNA damage compromises transcription in cells. Here we described a novel competitive transcription and adduct bypass (CTAB) assay to determine the effects of DNA lesions on the fidelity and efficiency of transcription. Using this strategy, we demonstrated that the oxidatively induced lesions 8,5′-cyclo-2′-deoxyadenosine (cdA) and 8,5′-cyclo-2′-deoxyguanosine (cdG), and methylglyoxal-induced N2-(1-carboxyethyl)-2′-deoxyguanosine (N2-CEdG) strongly inhibited transcription in vitro and in mammalian cells. In addition, cdA and cdG, but not N2-CEdG, induced transcriptional mutagenesis in vitro and in vivo. Furthermore, when located on the template DNA strand, all examined lesions were primarily repaired by transcription-coupled nucleotide excision repair (TC-NER) in mammalian cells. This newly developed CTAB assay should be generally applicable for quantitatively assessing how other DNA lesions impact DNA transcription in vitro and in cells.

Quantitative analysis of the mutagenic potential of 1-aminopyrene-DNA adduct bypass catalyzed by Y-family DNA polymerases

N-(Deoxyguanosin-8-yl)-1-aminopyrene (dGAP) is the predominant nitro polyaromatic hydrocarbon product generated from the air pollutant 1-nitropyrene reacting with DNA. Previous studies have shown that dGAP induces genetic mutations in bacterial and mammalian cells. One potential source of these mutations is the error-prone bypass of dGAP lesions catalyzed by the low-fidelity Y-family DNA polymerases. To provide a comparative analysis of the mutagenic potential of the translesion DNA synthesis (TLS) of dGAP, we employed short oligonucleotide sequencing assays (SOSAs) with the model Y-family DNA polymerase from Sulfolobus solfataricus, DNA Polymerase IV (Dpo4), and the human Y-family DNA polymerases eta (hPolη), kappa (hPolκ), and iota (hPolι). Relative to undamaged DNA, all four enzymes generated far more mutations (base deletions, insertions, and substitutions) with a DNA template containing a site-specifically placed dGAP. Opposite dGAP and at an immediate downstream template position, the most frequent mutations made by the three human enzymes were base deletions and the most frequent base substitutions were dAs for all enzymes. Based on the SOSA data, Dpo4 was the least error-prone Y-family DNA polymerase among the four enzymes during the TLS of dGAP. Among the three human Y-family enzymes, hPolκ made the fewest mutations at all template positions except opposite the lesion site. hPolκ was significantly less error-prone than hPolι and hPolη during the extension of dGAP bypass products. Interestingly, the most frequent mutations created by hPolι at all template positions were base deletions. Although hRev1, the fourth human Y-family enzyme, could not extend dGAP bypass products in our standing start assays, it preferentially incorporated dCTP opposite the bulky lesion. Collectively, these mutagenic profiles suggest that hPolk and hRev1 are the most suitable human Y-family DNA polymerases to perform TLS of dGAP in humans.

DNA Polymerase Eta Participates in the Mutagenic Bypass of Adducts Induced by Benzo[a]pyrene Diol Epoxide in Mammalian Cells

Y-family DNA-polymerases have larger active sites that can accommodate bulky DNA adducts allowing them to bypass these lesions during replication. One member, polymerase eta (pol eta), is specialized for the bypass of UV-induced thymidine-thymidine dimers, correctly inserting two adenines. Loss of pol eta function is the molecular basis for xeroderma pigmentosum (XP) variant where the accumulation of mutations results in a dramatic increase in UV-induced skin cancers. Less is known about the role of pol eta in the bypass of other DNA adducts. A commonly encountered DNA adduct is that caused by benzo[a]pyrene diol epoxide (BPDE), the ultimate carcinogenic metabolite of the environmental chemical benzo[a]pyrene. Here, treatment of pol eta-deficient fibroblasts from humans and mice with BPDE resulted in a significant decrease in Hprt gene mutations. These studies in mammalian cells support a number of in vitro reports that purified pol eta has error-prone activity on plasmids with site-directed BPDE adducts. Sequencing the Hprt gene from this work shows that the majority of mutations are G>T transversions. These data suggest that pol eta has error-prone activity when bypassing BPDE-adducts. Understanding the basis of environmental carcinogen-derived mutations may enable prevention strategies to reduce such mutations with the intent to reduce the number of environmentally relevant cancers.

Low Fidelity Bypass of O2-(3-Pyridyl)-4-oxobutylthymine, the Most Persistent Bulky Adduct Produced by the Tobacco Specific Nitrosamine 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone by Model DNA Polymerases

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the most important human carcinogens. It is metabolized to produce a variety of methyl and 4-(3-pyridyl)-4-oxo-butyl (POB) DNA adducts. A potentially important POB adduct is O(2)-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O(2)-POB-dT) because it is the most abundant POB adduct in NNK-treated rodents. To evaluate the mutagenic properties of O(2)-POB-dT, we measured the rate of insertion of dNTPs opposite and extension past both O(2)-POB-dT and O(2)-methylthymidine (O(2)-Me-dT) by two model polymerases, E. coli DNA polymerase I (Klenow fragment) with the proofreading exonuclease activity inactivated (Kf) and Sulfolobus solfataricus DNA polymerase IV (Dpo4). We found that the size of the alkyl chain only marginally affected the reactivity and that the specificity of adduct bypass was very low. The k(cat)/K(m) for the Kf catalyzed incorporation opposite and extension past the adducts was reduced ∼10(6)-fold when compared to undamaged DNA. Dpo4 catalyzed the incorporation opposite and extension past the adducts approximately 10(3)-fold more slowly than undamaged DNA. The dNTP specificity was less for Dpo4 than for Kf. In general, dA was the preferred base pair partner for O(2)-Me-dT and dT the preferred base pair partner for O(2)-POB-dT. With enzyme in excess over DNA, the time courses of the reactions showed a biphasic kinetics that indicates the formation inactive binary and ternary complexes.

Differential Roles for DNA Polymerases Eta, Zeta, and REV1 in Lesion Bypass of Intrastrand versus Interstrand DNA Cross-Links

Translesion DNA synthesis (TLS) is a process whereby specialized DNA polymerases are recruited to bypass DNA lesions that would otherwise stall high-fidelity polymerases. We provide evidence that TLS across cisplatin intrastrand cross-links is performed by multiple translesion DNA polymerases. First, we determined that PCNA monoubiquitination by RAD18 is necessary for efficient bypass of cisplatin adducts by the TLS polymerases eta (Poleta), REV1, and zeta (Polzeta) based on the observations that depletion of these proteins individually leads to decreased cell survival, cell cycle arrest in S phase, and activation of the DNA damage response. Second, we showed that in addition to PCNA monoubiquitination by RAD18, the Fanconi anemia core complex is also important for recruitment of REV1 to stalled replication forks in cisplatin treated cells. Third, we present evidence that REV1 and Polzeta are uniquely associated with protection against cisplatin and mitomycin C-induced chromosomal aberrations, and both are necessary for the timely resolution of DNA double-strand breaks associated with repair of DNA interstrand cross-links. Together, our findings indicate that REV1 and Polzeta facilitate repair of interstrand cross-links independently of PCNA monoubiquitination and Poleta, whereas RAD18 plus Poleta, REV1, and Polzeta are all necessary for replicative bypass of cisplatin intrastrand DNA cross-links.

DNA Polymerase: Structural Homology, Conformational Dynamics, and the Effects of Carcinogenic DNA Adducts

DNA replication is vital for an organism to proliferate and lying at the heart of this process is the enzyme DNA polymerase. Most DNA polymerases have a similar three dimensional fold, akin to a human right hand, despite differences in sequence homology. This structural homology would predict a relatively unvarying mechanism for DNA synthesis yet various polymerases exhibit markedly different properties on similar substrates, indicative of each type of polymerase being prescribed to a specific role in DNA replication. Several key conformational steps, discrete states, and structural moieties have been identified that contribute to the array of properties the polymerases exhibit. The ability of carcinogenic adducts to interfere with conformational processes by directly interacting with the protein explicates the mutagenic consequences these adducts impose. Recent studies have identified novel states that have been hypothesised to test the fit of the nascent base pair, and have also shown the enzyme to possess a lively quality by continually sampling various conformations. This review focuses on the homologous structural changes that take place in various DNA polymerases, both replicative and those involved in adduct bypass, the role these changes play in selection of a correct substrate, and how the presence of bulky carcinogenic adducts affects these changes.

Polymerization by DNA polymerase is blocked by cis-diamminedichloroplatinum(II) 1,3-d(GpTpG) cross-link: implications for cytotoxic effects in nucleotide excision repair-negative tumor cells

cis-Diamminedichloroplatinum(II) (cisplatin) forms DNA adducts that interfere with replication and transcription. The most common adducts formed in vivo are 1,2-intrastrand d(GpG) cross-links (Pt-GG) and d(ApG) cross-links (Pt-AG), with minor amounts of 1,3-d(GpNpG) cross-links (Pt-GNG), interstrand cross-links and monoadducts. Although the relative contribution of these different adducts to toxicity is not known, literature implicates that Pt-GG and Pt-AG adducts block replication. Thus, nucleotide excision repair (NER), by which platinum adducts are excised, and translesion DNA synthesis (TLS), which permits adduct bypass, are thought to be associated with cisplatin resistance. Recent studies have reported that the clinical benefit from platinum-based chemotherapy is high if tumor cells express low levels of NER factors. To investigate the role of platinum-DNA adducts in mediating tumor cell survival by TLS, we examined whether 1,3-intrastrand d(GpTpG) platinum cross-links (Pt-GTG), which probably exist in NER-negative tumor cells but not in NER-positive tumor cells, are bypassed by the translesion DNA polymerase eta (pol eta), which is known to bypass Pt-GG. We show that pol eta can incorporate the correct deoxycytidine triphosphate opposite the first 3'-cross-linked G of Pt-GTG but cannot insert any nucleotides opposite the second intact T or the third 5'-cross-linked G of the adducts, thereby suggesting that TLS does not facilitate replication past Pt-GTG adducts. Thus, our findings implicate Pt-GNG adducts as mediating the cytotoxicity of platinum-DNA adducts in NER-negative tumors in vivo.

Lesion Bypass of N2-Ethylguanine by Human DNA Polymerase ι

Nucleotide incorporation and extension opposite N2-ethyl-Gua by DNA polymerase iota was measured and structures of the DNA polymerase iota-N2-ethyl-Gua complex with incoming nucleotides were solved. Efficiency and fidelity of DNA polymerase iota opposite N2-ethyl-Gua was determined by steady state kinetic analysis with Mg2+ or Mn2+ as the activating metal. DNA polymerase iota incorporates dCMP opposite N2-ethyl-Gua and unadducted Gua with similar efficiencies in the presence of Mg2+ and with greater efficiencies in the presence of Mn2+. However, the fidelity of nucleotide incorporation by DNA polymerase iota opposite N2-ethyl-Gua and Gua using Mn2+ is lower relative to that using Mg2+ indicating a metal-dependent effect. DNA polymerase iota extends from the N2-ethyl-Gua:Cyt 3' terminus more efficiently than from the Gua:Cyt base pair. Together these kinetic data indicate that the DNA polymerase iota catalyzed reaction is well suited for N(2)-ethyl-Gua bypass. The structure of DNA polymerase iota with N2-ethyl-Gua at the active site reveals the adducted base in the syn configuration when the correct incoming nucleotide is present. Positioning of the ethyl adduct into the major groove removes potential steric overlap between the adducted template base and the incoming dCTP. Comparing structures of DNA polymerase iota complexed with N2-ethyl-Gua and Gua at the active site suggests movements in the DNA polymerase iota polymerase-associated domain to accommodate the adduct providing direct evidence that DNA polymerase iota efficiently replicates past a minor groove DNA adduct by positioning the adducted base in the syn configuration.