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

Abstract

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.

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

“…Polk is unique since it exists in the three biological domains, i.e., Eukarya, Archaea, and Bacteria, while other specialized Pols are present either in Eukarya or Bacterial/Archaea [Gerlach et al, 1999;Ogi et al, 1999;Wagner et al, 1999;Gruz et al, 2001]. Polk has been shown to bypass a variety of structurally unrelated DNA lesions such as N 2 -guanine adducts by benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) [Ogi et al, 2002;Suzuki et al, 2002;Huang et al, 2003;Avkin et al, 2004;Niimi et al, 2009;Sassa et al, 2011Sassa et al, , 2014Jha et al, 2016], 2-acetylaminofluorene [Zhang et al, 2000], methylglyoxal [Yuan et al, 2011], and monoalkylating agents [Choi et al, 2006], a C8-guanine adduct by 2amino-1-methyl-6-phenylimidazo[4,5-b]pyrimidine (PhIP) [Fukuda et al, 2009], thymine glycol [Fischhaber et al, 2002;Yoon et al, 2010], 8-oxoguanine [Zhang et al, 2000;Haracska et al, 2002;Jaloszynski et al, 2005], and interstrand cross-links [Minko et al, 2008;Takeiri et al, 2014;Kanemaru et al, 2017]. Besides TLS, Polk 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].…”

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

“…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.…”

“…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).…”

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

“…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).…”