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

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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.

Section: Introductionmentioning

confidence: 99%

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

Masumura

Toyoda-Hokaiwado

Niimi

et al. 2017

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“…The human pre-B cell line Nalm-6-MSH+ TK +/− (WT-MSH+) and the Polκ derivatives, KO and CD, were established as described previously (Suzuki et al 2013;Sassa et al 2014;Kanemaru et al 2015;Kanemaru et al 2017). Briefly, the KO cells (KO-MSH+) were established by deletion of exon 6 of the POLK gene where the catalytically essential amino acids D198 and E199 are present.…”

Section: Cell Lines and Culturementioning

confidence: 99%

“…Polκ is known to bypass an oxidative DNA lesion, that is, thymine glycol, in DNA accurately and efficiently (Yoon et al 2010). To examine the effects of Polκ on the practical threshold, we employed two human cell lines that either express catalytically dead Polκ (CD) or no Polκ (KO) along with the wild type Polκ (WT) cells (Sassa et al 2014;Kanemaru et al 2015;Kanemaru et al 2017). We employed not only KO cells but also CD cells because Polκ is known to interact with other proteins such as REV1, PCNA, or MSH2 (Ohashi et al 2009;Lv et al 2013;Yoon et al 2014a).…”

Section: Introductionmentioning

confidence: 99%

Effects of DNA polymerase kappa and mismatch repair on dose–responses of chromosome aberrations induced by three oxidative genotoxins in human cells

Nohmi

Matsumoto²

2019

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Genotoxic carcinogens are regulated under the policy that there is no threshold or safe dose. It has been pointed out, however, that self-defense mechanisms, such as detoxification, DNA repair, and error-free translesion synthesis, may protect chromosome DNA from genotoxic insults, thereby constituting practical threshold. In this study, we examined dose responses of chromosome aberrations induced by three oxidative genotoxins, that is, hydrogen peroxide (H 2 O 2), menadione and paraquat, with or without DNA polymerase kappa (Polκ) activities and mismatch repair capacities in human cells. Polκ is involved in translesion synthesis across DNA damage and mismatch repair is responsible for correction of base-base mismatch in DNA. Polκ activity of the cells was inactivated either by point mutations in the catalytically essential amino acids (catalytically dead or CD) or by deletion of the POLK gene (knockout or KO). In the absence of mismatch repair, frequencies of chromosome aberrations induced by H 2 O 2 and menadione were not significantly different among CD, KO, and the wild type (WT) cells. In the presence of mismatch repair, however, cytotoxicity and clastogenicity were enhanced and Polκ modulated the sensitivity of the cells. No-observed-genotoxiceffect-levels (NOGELs) for H 2 O 2 and menadione were CD = KO < WT cells. In contrast, the sensitivities of the cells to paraquat were not significantly affected by the status of mismatch repair or Polκ activity. The results suggest that mismatch repair and Polκ coordinately modulate NOGELs for the clastogenicity of H 2 O 2 and menadione and also that DNA lesion(s) responsible for paraquat-induced chromosome aberrations are different from those induced by H 2 O 2 and menadione.

“…Polκ is unique in that its homologues are present in three domains of life, Bacteria, Archaea and Eukarya (Wagner et al, 1999;Gerlach et al, 2001;Gruz et al, 2001). Previous biochemical and cultured cell studies reveal that Polκ bypasses a variety of structurally diverse lesions in DNA, including N 2 -2 0deoxyguanosine (N 2 -dG) adducts by active metabolites of BP, i.e., BP-7,8-dihydrodiol-9,10-epoxide (BPDE) (Ogi et al, 2002;Suzuki et al, 2002;Avkin et al, 2004;Niimi et al, 2009;Kanemaru et al, 2015), monoalkylating agents (Choi et al, 2006) and methylglyoxal (Yuan et al, 2011), a C8-guanine adduct by 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyrimidine (PhIP) (Fukuda et al, 2009), thymine glycol (Fischhaber et al, 2002), 8-oxo-7,8-dihydro-dG (8-oxo-dG) (Zhang et al, 2000;Kamiya and Kurokawa, 2012) and intrastrand crosslinks (Minko et al, 2008;Takeiri et al, 2014;Kanemaru et al, 2017). In addition, Polκ appears to be involved in nucleotide excision repair (Ogi et al, 2010), replication checkpoint (Betous et al, 2013), DNA synthesis through non-B structures (Betous et al, 2009) and repair of strand breaks in DNA (Zhang et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

DNA polymerase kappa counteracts inflammation‐induced mutagenesis in multiple organs of mice

Hakura

Sui

Sonoda

et al. 2019

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In vitro studies indicate that DNA polymerase kappa (Polκ) is able to accurately and efficiently perform DNA synthesis using templates containing various types of DNA damage, including benzo[a]pyrene (BP)‐induced N2‐deoxyguanosine adducts. In this study, we examined sensitivity of inactivated Polk knock‐in (Polk−/−) mice to BP carcinogenicity in the colon by administering an oral dose of BP plus dextran sulfate sodium (DSS), an inflammation causing promoter of carcinogenesis. Although colon cancer was successfully induced by BP plus DSS, there was no significant difference in tumor incidence or multiplicity between Polk−/− and Polk+/+ mice. Malignant lymphoma was induced in thymus by the treatment only in Polk−/− mice, but it lacked statistical significance. Mutant frequencies (MFs) in the gpt reporter gene were strongly enhanced in colon; almost to the same extent in both types of mice. Micronucleus formation in bone marrow at the high dose of BP and DNA adducts in colon and lung was not significantly different between two types of mice. Surprisingly, however, Polk−/− mice exhibited significantly higher MFs in colon and lung than did Polk+/+ mice when they were treated with DSS alone. The most prominent mutation induced by DSS treatment was G:C to C:G transversion, whose specific MF in proximal colon was 30 times higher in Polk−/− than in Polk+/+ mice. DSS alone did not enhance MF at all in Polk+/+ mice. The results indicate that Polκ does not suppress BP‐induced mutagenesis and carcinogenesis in the colon, but counteracts inflammation‐induced mutagenesis in multiple organs. Environ. Mol. Mutagen. 60:320–330, 2019. © 2019 Wiley Periodicals, Inc.