In vivo evidence that phenylalanine 171 acts as a molecular brake for translesion DNA synthesis across benzo[a]pyrene DNA adducts by human DNA polymerase κ

Sassa, Akira; Suzuki, Tetsuya; Kanemaru, Yuki; Niimi, Naoko; Fujimoto, Haruhiro; Katafuchi, Atsushi; Grúz, Petr; Yasui, Masayoshi; Gupta, Ramesh C.; Johnson, Francis; Ohta, Toshihiro; Honma, Masamitsu; Adachi, Noritaka; Nohmi, Takehiko

doi:10.1016/j.dnarep.2013.12.008

Cited by 10 publications

(22 citation statements)

References 57 publications

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“…1d). The enhanced mutagenicity of BPDE in Pol κ-deficient cells is consistent with previous studies using the Hprt gene mutation assay in mouse ES cells [8] and the supF forward mutation assay in the parental Nalm-6 human Pre-B cell line [25]. Therefore, we judged the TK gene mutation assay in the established cell lines to be functional for analyzing the genotoxicity of test articles.…”

Section: Resultssupporting

confidence: 86%

“…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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Section: Resultssupporting

confidence: 86%

Section: Methodsmentioning

confidence: 99%

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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“…After homogenization of the Polk mutant allele, C57BL6-Polk tm3(mPolk)Csk mice, referred to in this study as Polk D197A-E198A mice, were established. The Western blotting for the expression of D197A-E198A and wild-type Polk in Polk D197A-E198A mice and littermate wild-type mice, respectively, was conducted with polyclonal antisera against human Polk [21].…”

Section: Animalsmentioning

confidence: 99%

“…Polk is unique in that its orthologs are present in Eukarya, bacteria and Archaea [15][16][17]. Several lines of in vitro evidence suggest that Polk may be involved in TLS across a variety of DNA damage, such as N 2 -guanyl adducts induced by polycyclic aromatic hydrocarbons and alkylating agents [18][19][20][21][22][23], a C8-guanyl adduct by 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) [24], thymine glycol [25,26], 8-oxo-guanine [27,28], and interstrand DNA cross-links [29]. In addition to TLS, Polk is reported to be involved in nucleotide excision repair [30], replication checkpoint [31], repair of single-strand breaks in DNA [32], and microsatellite stability [33].…”

Section: Introductionmentioning

confidence: 98%

In vivo evidence that DNA polymerase kappa is responsible for error-free bypass across DNA cross-links induced by mitomycin C

Takeiri¹,

Wada²,

Motoyama³

et al. 2014

DNA Repair

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

Kanemaru

Suzuki

Niimi

et al. 2015

Environ and Mol Mutagen

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DNA polymerase κ (Pol κ) is a specialized DNA polymerase involved in translesion DNA synthesis. Although its bypass activities across lesions are well characterized in biochemistry, its cellular protective roles against genotoxic insults are still elusive. To better understand the in vivo protective roles, we have established a human cell line deficient in the expression of Pol κ (KO) and another expressing catalytically dead Pol κ (CD), to examine the cytotoxic sensitivity to 11 genotoxins including ultraviolet C light (UV). These cell lines were established in a genetic background of Nalm‐6‐MSH+, a human lymphoblastic cell line that has high efficiency for gene targeting, and functional p53 and mismatch repair activities. We classified the genotoxins into four groups. Group 1 includes benzo[a]pyrene diolepoxide, mitomycin C, and bleomycin, where the sensitivity was equally higher in KO and CD than in the cell line expressing wild‐type Pol κ (WT). Group 2 includes hydrogen peroxide and menadione, where hypersensitivity was observed only in KO. Group 3 includes methyl methanesulfonate and ethyl methanesulfonate, where hypersensitivity was observed only in CD. Group 4 includes UV and three chemicals, where the chemicals exhibited similar cytotoxicity to all three cell lines. The results suggest that Pol κ not only protects cells from genotoxic DNA lesions via DNA polymerase activities, but also contributes to genome integrity by acting as a non‐catalytic protein against oxidative damage caused by hydrogen peroxide and menadione. The non‐catalytic roles of Pol κ in protection against oxidative damage by hydrogen peroxide are discussed. Environ. Mol. Mutagen. 56:650–662, 2015. © 2015 Wiley Periodicals, Inc.

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In vivo evidence that phenylalanine 171 acts as a molecular brake for translesion DNA synthesis across benzo[a]pyrene DNA adducts by human DNA polymerase κ

Cited by 10 publications

References 57 publications

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

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

In vivo evidence that DNA polymerase kappa is responsible for error-free bypass across DNA cross-links induced by mitomycin C

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

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