Efficiency and Accuracy of SOS-induced DNA Polymerases Replicating Benzo[a]pyrene-7,8-diol 9,10-Epoxide A and G Adducts

Shen, Xuan; Sayer, Jane M.; Kroth, Heiko; Pontén, Ingrid; O’Donnell, Michael; Woodgate, Roger; Jerina, Donald M.; Goodman, Myron F.

doi:10.1074/jbc.m109575200

Cited by 115 publications

(113 citation statements)

References 57 publications

(66 reference statements)

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“…Similar results were obtained with plasmid GP-BPG2 (kan R ) (52 Ϯ 4%; Table I (top)). This extent of bypass is much higher than values usually reported for bypass across BP-G adducts in E. coli (37)(38)(39). However, since the differences might stem from DNA sequence context effects, which are known to strongly affect bypass across BP-G adducts in vitro and in E. coli in vivo (40 -43), we assayed TLS across the BP-G adduct in E. coli cells, using the same gapped plasmids used in the mammalian cells.…”

Section: Tls Across a Benzo[a]pyrene-guanine Adduct Is More Efficientmentioning

confidence: 84%

Quantitative Analysis of Translesion DNA Synthesis across a Benzo[a]pyrene-Guanine Adduct in Mammalian Cells

Avkin

Goldsmith

Velasco-Miguel

et al. 2004

Journal of Biological Chemistry

172

177

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Replication across unrepaired DNA lesions in mammalian cells is effected primarily by specialized, low fidelity DNA polymerases. We studied translesion DNA synthesis (TLS) across a benzo[a]pyrene-guanine (BP-G) adduct, a major mutagenic DNA lesion generated by tobacco smoke. This was done using a quantitative assay that measures TLS indirectly, by measuring the recovery of gapped plasmids transfected into cultured mammalian cells. Analysis of PolK ؉/؉ mouse embryo fibroblasts (MEFs) showed that TLS across the BP-G adduct occurred with an efficiency of 48 ؎ 4%, which is an order of magnitude higher than in Escherichia coli. In PolK ؊/؊ MEFs, bypass was 16 ؎ 1%, suggesting that at least twothirds of the BP-G adducts in MEFs were bypassed exclusively by polymerase (pol ). In contrast, pol was not required for bypass across BP-G in a human XP-V cell line. Analysis of misinsertion specificity across BP-G revealed that bypass was more error-prone in MEFs lacking pol . Expression of pol from a plasmid introduced into PolK ؊/؊ MEFs restored both the extent and fidelity of bypass across BP-G. Pol was not required for bypass of a synthetic abasic site. In vitro analysis demonstrated efficient bypass across BP-G by both pol and pol , suggesting that the biological role of pol in TLS across BP-G is due to regulation of TLS and not due to an exclusive ability to bypass this lesion. These results indicate that BP-G is bypassed in mammalian cells with relatively high efficiency and that pol bypasses BP-G in vivo with higher efficiency and higher accuracy than other DNA polymerases.Genomic DNA is constantly subject to damage caused by both external agents, such as sunlight, and endogenous chemicals, such as reactive oxygen species. Most of this damage is eliminated by error-free DNA repair mechanisms, thereby restoring the DNA to its native sequence (1). However, a significant number of lesions escape repair and might therefore interfere with DNA replication and gene expression. Such interference can be mitigated by DNA damage tolerance mechanisms, primarily translesion DNA synthesis (TLS 1 ; also termed translesion replication) (2-5) and postreplicative recombinational repair (1, 6 -8). The key components in TLS are low fidelity DNA polymerases that specialize in lesion bypass (9 -12). These proteins were conserved in evolution and are present in organisms ranging from Escherichia coli to humans (13). Humans contain at least four specialized DNA polymerases belonging to the Y superfamily (pol , pol , pol , and REV1) as well as several from other polymerase families (e.g. pol (14), pol (15, 16), and pol (16, 17)). Many of these polymerases have been implicated in TLS in vitro (18 -24). However, there is a paucity of information about the efficiency and fidelity with which they support lesion bypass in living cells.Pol has a well established biological role in TLS, since it is mutated in all patients examined with the variant form of the hereditary disease xeroderma pigmentosum (10,18). This disease is characterized by sensitivi...

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Section: Tls Across a Benzo[a]pyrene-guanine Adduct Is More Efficientmentioning

confidence: 84%

Quantitative Analysis of Translesion DNA Synthesis across a Benzo[a]pyrene-Guanine Adduct in Mammalian Cells

Avkin

Goldsmith

Velasco-Miguel

et al. 2004

Journal of Biological Chemistry

172

177

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“…Polκ is thought to be involved in the elongation step following mismatched bases or following damaged bases [110][111][112], and reads through bulky adducts such as modifications by benzo[a]pyrene diol epoxide (BPDE) [113,114]. When nucleotides containing dG-N 2 -BPDE, the most potent carcinogenic compound produced by industrial and cigarette smoke, are used as template, Polκ can bypass the adduct with much higher efficiency than Polη or Polι by correctly inserting C opposite the bulky lesion [114].…”

Section: Y Family Dna Polymerasesmentioning

confidence: 99%

Eukaryotic DNA damage tolerance and translesion synthesis through covalent modifications of PCNA

Andersen¹,

Xiao³

2007

Cell Res

183

187

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npg In addition to well-defined DNA repair pathways, all living organisms have evolved mechanisms to avoid cell death caused by replication fork collapse at a site where replication is blocked due to disruptive covalent modifications of DNA. The term DNA damage tolerance (DDT) has been employed loosely to include a collection of mechanisms by which cells survive replication-blocking lesions with or without associated genomic instability. Recent genetic analyses indicate that DDT in eukaryotes, from yeast to human, consists of two parallel pathways with one being error-free and another highly mutagenic. Interestingly, in budding yeast, these two pathways are mediated by sequential modifications of the proliferating cell nuclear antigen (PCNA) by two ubiquitination complexes Rad6-Rad18 and Mms2-Ubc13-Rad5. Damage-induced monoubiquitination of PCNA by Rad6-Rad18 promotes translesion synthesis (TLS) with increased mutagenesis, while subsequent polyubiquitination of PCNA at the same K164 residue by Mms2-Ubc13-Rad5 promotes error-free lesion bypass. Data obtained from recent studies suggest that the above mechanisms are conserved in higher eukaryotes. In particular, mammals contain multiple specialized TLS polymerases. Defects in one of the TLS polymerases have been linked to genomic instability and cancer. DNA damage toleranceIn the presence of spontaneous or carcinogen-induced DNA damage, living cells have to maintain and complete DNA synthesis or risk replication fork collapse. Since the process of DNA licensing is to ensure the genome is duplicated once and only once during each cell cycle, stalled or collapsed replication forks may not be able to restart, which often results in double-strand breaks (DSBs) and causes compromised genome integrity or cell death. In addition to highly conserved DNA repair pathways, all living organisms have evolved schemes to ensure continuation of DNA synthesis in the presence of damage. These schemes were originally termed DNA postreplication repair (PRR) due to observations of transient shortened nascent DNA structures following S phase in response to DNA damage. In bacteria and unicellular yeast, these shortened DNA segments can be measured by an alkaline sedimentation assay [1] or directly observed in electron micrographs [2]. In wild-type cells, these truncated DNA segments were restored to full length following a short recovery time. One typical experiment [1] involved the restoration of the nascent strand following UV exposure in nucleotide excision repair (NER)-deficient cells and was originally assumed to represent a mechanism of DNA repair. However, further investigation revealed that, although the nascent fragments were re-annealed, the original UV-induced pyrimidine dimers, which were responsible for the generation of single-strand gaps, often persisted in the genome [3,4]. It was argued that the replication-blocking lesion was not necessarily corrected, but rather transiently bypassed and carried over to the next generation. Perhaps it is more beneficial for the organi...

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“…E. coli DinB can also correctly incorporate dCMP opposite some N 2 -dG adducts, including N 2 -furfuryl-dG (15) and N 2 -(1-carboxyethyl)-2′-dG (16) with greater catalytic proficiency than opposite normal dG. Similarly, BPDE-dG (15,17) is incorporated with high accuracy but reduced efficiency. Although phylogenetically related to Polκ, the enzyme Dpo4 possesses UV-lesion bypass activity akin to that of Polη (18), but bypasses BPDE-dG lesions inefficiently and induces mutations by a −1 frame-shift mechanism (19).…”

mentioning

confidence: 99%

Variants of mouse DNA polymerase κ reveal a mechanism of efficient and accurate translesion synthesis past a benzo[ a ]pyrene dG adduct

Liu

Yang

Tang

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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DNA polymerase κ (Polκ) is the only known Y-family DNA polymerase that bypasses the 10S (+)-trans-anti-benzo[a]pyrene diol epoxide (BPDE)-N 2 -deoxyguanine adducts efficiently and accurately. The unique features of Polκ, a large structure gap between the catalytic core and little finger domain and a 90-residue addition at the N terminus known as the N-clasp, may give rise to its special translesion capability. We designed and constructed two mouse Polκ variants, which have reduced gap size on both sides [Polκ Gap Mutant (PGM) 1] or one side flanking the template base (PGM2). These Polκ variants are nearly as efficient as WT in normal DNA synthesis, albeit with reduced accuracy. However, PGM1 is strongly blocked by the 10S (+)-trans-anti-BPDE-N 2 -dG lesion. Steady-state kinetic measurements reveal a significant reduction in efficiency of dCTP incorporation opposite the lesion by PGM1 and a moderate reduction by PGM2. Consistently, Polκ-deficient cells stably complemented with PGM1 GFP-Polκ remained hypersensitive to BPDE treatment, and complementation with WT or PGM2 GFP-Polκ restored BPDE resistance. Furthermore, deletion of the first 51 residues of the N-clasp in mouse Polκ (mPolκ 52-516 ) leads to reduced polymerization activity, and the mutant PGM2 52-516 but not PGM1 52-516 can partially compensate the N-terminal deletion and restore the catalytic activity on normal DNA. However, neither WT nor PGM2 mPolκ 52-516 retains BPDE bypass activity. We conclude that the structural gap physically accommodates the bulky aromatic adduct and the N-clasp is essential for the structural integrity and flexibility of Polκ during translesion synthesis.translesion DNA synthesis | polycyclic aromatic hydrocarbons B enzo[a]pyrene (BP) is one of the best characterized polycyclic aromatic hydrocarbons generated during incomplete fuel combustion, in tobacco smoke, and in cooked food. The most mutagenic and tumorigenic metabolite of BP in vivo is the (+)-7R,8S,9S,10R-BP dihydrodiol epoxide [(+)-anti-BPDE] (1), which reacts readily with the exocyclic amino groups of guanine residues in DNA to form the major 10S (+)-trans-anti-BPDE-N 2 -dG adduct (2) (abbreviated as BPDE-dG hereafter). This adduct typically impedes normal DNA replication. Translesion DNA synthesis (TLS), which provides one mode of DNA damage tolerance, uses specialized bypass polymerases to synthesize DNA opposite and beyond a variety of replication-blocking lesions, thus avoiding replication fork collapse (3). The Y-family DNA polymerases are specialized for TLS. To date, four of them, namely DNA polymerase (Pol) η, Polι, Polκ, and REV1, have been identified in mammals (4-6). Polκ is the only one with homologs in bacteria (DinB; also known as Pol IV in Escherichia coli), archaea [DNA polymerase IV (Dpo4) in Sulfolobus solfataricus and DinB homologue (Dbh) in Sulfolobus acidocaldarius] and all eukaryotes. The mouse and human POLK gene are expressed ubiquitously, with highest levels in testis (7).Human Polκ can accurately incorporate dCMP opposite the BPDE-dG adduct ...

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Efficiency and Accuracy of SOS-induced DNA Polymerases Replicating Benzo[a]pyrene-7,8-diol 9,10-Epoxide A and G Adducts

Cited by 115 publications

References 57 publications

Quantitative Analysis of Translesion DNA Synthesis across a Benzo[a]pyrene-Guanine Adduct in Mammalian Cells

Quantitative Analysis of Translesion DNA Synthesis across a Benzo[a]pyrene-Guanine Adduct in Mammalian Cells

Eukaryotic DNA damage tolerance and translesion synthesis through covalent modifications of PCNA

Variants of mouse DNA polymerase κ reveal a mechanism of efficient and accurate translesion synthesis past a benzo[ a ]pyrene dG adduct

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