Catalytic and non‐catalytic roles of <scp>DNA</scp> polymerase κ in the protection of human cells against genotoxic stresses

Kanemaru, Yuki; Suzuki, Tetsuya; Niimi, Naoko; Grúz, Petr; Matsumoto, Kyomu; Adachi, Noritaka; Honma, Masamitsu; Nohmi, Takehiko

doi:10.1002/em.21961

Cited by 22 publications

(34 citation statements)

References 71 publications

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“…In fact, although both the POLK knock-out cells and the inactivated POLK knock-in cells show equal hypersensitivity to benzo[ a ]pyrene and mitomycin C, the knock-out cells show hypersensitivity to oxidizing agents but not alkylating agents, whereas the latter cells show exactly the opposite, suggesting complex functions of inactive pol κ against different genotoxic stresses, i.e., a non-catalytic protective role against oxidative damage but a dominant negative deleterious effect on methylation damage repair. 49 In this perspective, it would be desirable to carry out further investigations to delineate in vivo impacts of those dysfunctional pol κ variants at the cellular and organismal levels. It is also plausible that pol κ variants to alter protein interactions may significantly change their TLS efficiency in cells, in that the protein-protein interactions of Y-family pols have been suggested to be involved in regulation of cellular TLS processes.…”

Section: Discussionmentioning

confidence: 99%

Six Germline Genetic Variations Impair the Translesion Synthesis Activity of Human DNA Polymerase κ

Kim

Yeom

Hong

et al. 2016

Chem. Res. Toxicol.

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DNA polymerase (pol) κ efficiently catalyzes error-free translesion DNA synthesis (TLS) opposite bulky N2-guanyl lesions induced by carcinogens such as polycyclic aromatic hydrocarbons. We investigated the biochemical effects of nine human nonsynonymous germline POLK variations on the TLS properties of pol κ, utilizing recombinant pol κ (residues 1–526) enzymes and DNA templates containing an N2-CH2(9-anthracenyl)G (N2-AnthG), 8-oxo-7,8-dihydroguanine (8-oxoG), O6-methyl(Me)G, or an abasic site. In steady-state kinetic analyses, the R246X, R298H, T473A, and R512W variants displayed 7- to 18-fold decreases in kcat/Km for dCTP insertion opposite G and N2-AnthG, with 2- to 3-fold decreases in DNA binding affinity, compared to wild-type, and further showed 5- to 190-fold decreases in kcat/Km for next-base extension from C paired with N2-AnthG. The A471V variant showed 2- to 4-fold decreases in kcat/Km for correct nucleotide insertion opposite and beyond G (or N2-AnthG) compared to wild-type. These five hypoactive variants also showed similar patterns of attenuation of TLS activity opposite 8-oxoG, O6-MeG, and abasic lesions. By contrast, the T44M variant exhibited 7- to 11-fold decreases in kcat/Km for dCTP insertion opposite N2-AnthG and O6-MeG (as well as for dATP insertion opposite an abasic site), but not opposite both G and 8-oxoG, nor beyond N2-AnthG, compared to wild-type. These results suggest that the R246X, R298H, T473A, R512W, and A471V variants cause a general catalytic impairment of pol κ opposite G and all four lesions, whereas the T44M variant induces opposite lesion-dependent catalytic impairment—i.e., only opposite O6-MeG, abasic, and bulky N2-G lesions, but not opposite G and 8-oxoG—in pol κ, which might indicate that these hypoactive pol κ variants are genetic factors in modifying individual susceptibility to genotoxic carcinogens in certain subsets of populations.

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Section: Discussionmentioning

confidence: 99%

Six Germline Genetic Variations Impair the Translesion Synthesis Activity of Human DNA Polymerase κ

Kim

Yeom

Hong

et al. 2016

Chem. Res. Toxicol.

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“…We did not utilize CD TK+/- cells, as KO and CD cells exhibited similar sensitivity to MMC treatment in our previous study [26]. Results from the TK gene mutation assay revealed a higher mutant frequency after MMC treatment in KO TK+/- cells than in WT TK+/- cells (Fig.…”

Section: Discussionmentioning

confidence: 94%

“…The resulting cell lines were named as WT TK+/-, KO TK+/-, and CD TK+/- based on the different expression profile of Pol κ. Those three cell lines had comparable doubling times (WT TK+/-: 21.5 ± 0.6 h, KO TK+/-: 20.9 ± 0.6 h, and CD TK+/-: 22.2 ± 0.8 h), which were not significantly different from the original WT, KO, and CD cells, respectively [26]. The spontaneous TK mutant frequency of KO TK+/- cells was higher than those of WT TK+/- and CD TK+/- cells, but this difference was not statistically significant (Table 1).…”

Section: Resultsmentioning

confidence: 99%

“…The human pre-B cell line Nalm-6-MSH+ (WT) and its Pol κ derivatives, KO and CD cells, were established in our laboratory as described previously [25, 26, 34]. The KO cells were generated by deleting exon 6 of the POLK , resulting in a frameshift.…”

Section: Methodsmentioning

confidence: 99%

“…To better understand the protective roles of Pol κ at the cellular level, we previously generated human Pol κ knockout (KO) [25] and catalytically dead (CD) mutants in the human Nalm-6-MSH+ cell line and examined cytotoxic sensitivity to various genotoxins [26]. We conducted the study with not only KO cells but also CD cells because Pol κ interacts with other proteins, such as REV1 and proliferating cell nuclear antigen PCNA.…”

Section: Introductionmentioning

confidence: 99%

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DNA polymerase kappa protects human cells against MMC-induced genotoxicity through error-free translesion DNA synthesis

et al. 2017

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BackgroundInteractions between genes and environment are critical factors for causing cancer in humans. The genotoxicity of environmental chemicals can be enhanced via the modulation of susceptible genes in host human cells. DNA polymerase kappa (Pol κ) is a specialized DNA polymerase that plays an important role in DNA damage tolerance through translesion DNA synthesis. To better understand the protective roles of Pol κ, we previously engineered two human cell lines either deficient in expression of Pol κ (KO) or expressing catalytically dead Pol κ (CD) in Nalm-6-MSH+ cells and examined cytotoxic sensitivity against various genotoxins. In this study, we set up several genotoxicity assays with cell lines possessing altered Pol κ activities and investigated the protective roles of Pol κ in terms of genotoxicity induced by mitomycin C (MMC), a therapeutic agent that induces bulky DNA adducts and crosslinks in DNA.ResultsWe introduced a frameshift mutation in one allele of the thymidine kinase (TK) gene of the KO, CD, and wild-type Pol κ cells (WT), thereby establishing cell lines for the TK gene mutation assay, namely TK+/- cells. In addition, we formulated experimental conditions to conduct chromosome aberration (CA) and sister chromatid exchange (SCE) assays with cells. By using the WT TK+/- and KO TK+/- cells, we assayed genotoxicity of MMC. In the TK gene mutation assay, the cytotoxic and mutagenic sensitivities of KO TK+/- cells were higher than those of WT TK+/- cells. MMC induced loss of heterozygosity (LOH), base pair substitutions at CpG sites and tandem mutations at GpG sites in both cell lines. However, the frequencies of LOH and base substitutions at CpG sites were significantly higher in KO TK+/- cells than in WT TK+/- cells. MMC also induced CA and SCE in both cell lines. The KO TK+/- cells displayed higher sensitivity than that displayed by WT TK+/- cells in the SCE assay.ConclusionsThese results suggest that Pol κ is a modulating factor for the genotoxicity of MMC and also that the established cell lines are useful for evaluating the genotoxicity of chemicals from multiple endpoints in different genetic backgrounds of Pol κ.Electronic supplementary materialThe online version of this article (doi:10.1186/s41021-016-0067-3) contains supplementary material, which is available to authorized users.

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Limited ability of DNA polymerase kappa to suppress benzo[a]pyrene‐induced genotoxicity in vivo

Masumura

Toyoda-Hokaiwado

Niimi

et al. 2017

Environ and Mol Mutagen

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DNA polymerase kappa (Polk) is a specialized DNA polymerase involved in translesion DNA synthesis. To understand the protective roles against genotoxins in vivo, we established inactivated Polk knock-in gpt delta (inactivated Polk KI) mice that possessed reporter genes for mutations and expressed inactive Polk. In this study, we examined genotoxicity of benzo[a]pyrene (BP) to determine whether Polk actually suppressed BP-induced genotoxicity as predicted by biochemistry and in vitro cell culture studies. Seven-week-old inactivated Polk KI and wild-type (WT) mice were treated with BP at doses of 5, 15, or 50 mg/(kg·day) for three consecutive days by intragastric gavage, and mutations in the colon and micronucleus formation in the peripheral blood were examined. Surprisingly, no differences were observed in the frequencies of mutations and micronucleus formation at 5 or 50 mg/kg doses. Inactivated Polk KI mice exhibited approximately two times higher gpt mutant frequency than did WT mice only at the 15 mg/kg dose. The frequency of micronucleus formation was slightly higher in inactivated Polk KI than in WT mice at the same dose, but it was statistically insignificant. The results suggest that Polk has a limited ability to suppress BP-induced genotoxicity in the colon and bone marrow and also that the roles of specialized DNA polymerases in mutagenesis and carcinogenesis should be examined not only by in vitro assays but also by in vivo mouse studies. We also report the spontaneous mutagenesis in inactivated Polk KI mice at young and old ages. Environ. Mol. Mutagen. 58:644-653, 2017. © 2017 Wiley Periodicals, Inc.

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Catalytic and non‐catalytic roles of DNA polymerase κ in the protection of human cells against genotoxic stresses

Cited by 22 publications

References 71 publications

Six Germline Genetic Variations Impair the Translesion Synthesis Activity of Human DNA Polymerase κ

Six Germline Genetic Variations Impair the Translesion Synthesis Activity of Human DNA Polymerase κ

DNA polymerase kappa protects human cells against MMC-induced genotoxicity through error-free translesion DNA synthesis

Limited ability of DNA polymerase kappa to suppress benzo[a]pyrene‐induced genotoxicity in vivo

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