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

Takeiri, Akira; Wada, Naoko; Motoyama, Shigeki; Matsuzaki, Kaori; Tateishi, Hiromi; Matsumoto, Kaoru; Niimi, Naoko; Sassa, Akira; Grúz, Petr; Masumura, Ken-ichi; Yamada, Masami; Mishima, Masayuki; Jishage, Kou-ichi; Nohmi, Takehiko

doi:10.1016/j.dnarep.2014.09.002

Cited by 18 publications

(27 citation statements)

References 60 publications

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“…Recently, it has been reported that gpt delta mice expressing catalytically inactive Pol κ exhibited an elevated frequency of mutations at CpG and also GpG sequences in bone marrow after MMC treatment [32]. The results presented here with CpG inter-strand crosslinks are consistent with the in vivo results and are also supported by biochemical evidence that Pol κ accurately bypasses the N 2 - N 2 inter-strand crosslinks at CpG sites [19].…”

Section: Discussionsupporting

confidence: 88%

“…Although the amounts of DNA inter-strand crosslinks are lower than those of intra-strand crosslinks or monofunctional adducts, these inter-strand crosslinks are largely responsible for the cytotoxicity of MMC [31]. Earlier studies reported that Pol κ is involved in error-free TLS across N 2 -guanine inter-strand crosslinks and N 2 -guanine monofunctional adducts [19, 32, 33]. However, the roles Pol κ plays to protect cells from mutagenic and clastogenic effects of multiple DNA damages induced by MMC in human cells are unclear.…”

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: Discussionsupporting

confidence: 88%

Section: Introductionmentioning

confidence: 99%

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

et al. 2017

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“…22 Recently, using a knock-in mouse model that express catalytically inactive pol κ , Takeiri et al reported in vivo evidence that pol κ is responsible for error-free bypass across ICLs induced by mitomycin C (a type of N 2 -G-N 2 -G ICL). 59 In contrast, only moderate bypass activities of pol κ were observed in vitro toward N7-G-N7-G (major groove lesions) ICLs derived from cisplatin and nitrogen mustard. 21 Therefore, given that DOB-ICL is a major groove lesion, it is not surprising that pol κ showed low bypass activity.…”

Section: Discussionmentioning

confidence: 99%

Mutagenic Bypass of an Oxidized Abasic Lesion-Induced DNA Interstrand Cross-Link Analogue by Human Translesion Synthesis DNA Polymerases

Xu¹,

Ouellette²,

Ghosh

et al. 2015

Biochemistry

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5′-(2-Phosphoryl-1,4-dioxobutane) (DOB) is an oxidized abasic site that is produced by several antitumor agents and γ-radiolysis. DOB reacts reversibly with a dA opposite the 3′-adjacent nucleotide to form DNA interstrand cross-links (ICLs), genotoxic DNA lesions that can block DNA replication and transcription. Translesion synthesis (TLS) is an important step in several ICL repair pathways to bypass unhooked intermediates generated by endonucleolytic incision. The instability of DOB-ICLs has made it difficult to learn about their TLS-mediated repair capability and mutagenic potential. We recently developed a method for chemically synthesizing oligonucleotides containing a modified DOB-ICL analogue. Herein, we examined the capabilities of several highly relevant eukaryotic TLS DNA polymerases (pols), including human pol η, pol κ, pol ι, pol ν, REV1, and yeast pol ζ, to bypass this DOB-ICL analogue. The prelesion, translesion, and postlesion replication efficiency and fidelity were examined. Pol η showed moderate bypass activity when encountering the DOB-ICL, giving major products one or two nucleotides beyond the cross-linked template nucleotide. In contrast, DNA synthesis by the other pols was stalled at the position before the cross-linked nucleotide. Steady-state kinetic data and liquid chromatography–mass spectrometry sequencing of primer extension products by pol η unambiguously revealed that pol η-mediated bypass is highly error-prone. Together, our study provides the first set of in vitro evidence that the DOB-ICL is a replication-blocking and highly miscoding lesion. Compared to several other TLS pols examined, pol η is likely to contribute to the TLS-mediated repair of the DOB-ICL in vivo.

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“…Consistent with this property, Pol κ−/− cells are sensitive to exposure to MMC, which forms ICLs in the minor groove [18, 30, 59, 60]. Interestingly, Pol κ −/− cells were found to be more sensitive to both MMC and cisplatin in the G0/G1 phase of the cell cycle, suggesting a more important role for Pol κ in replication-independent repair processes [30].…”

Section: Tls Polymerases In Icl Repairmentioning

confidence: 95%

Involvement of translesion synthesis DNA polymerases in DNA interstrand crosslink repair

Roy

Schärer

2016

DNA Repair

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DNA interstrand crosslinks (ICLs) covalently join the two strands of a DNA duplex and block essential processes such as DNA replication and transcription. Several important anti-tumor drugs such as cisplatin and nitrogen mustards exert their cytotoxicity by forming ICLs. However, multiple complex pathways repair ICLs and these are thought to contribute to the development of resistance towards ICL-inducing agents. While the understanding of many aspects of ICL repair is still rudimentary, studies in recent years have provided significant insights into the pathways of ICL repair. In this perspective we review the recent advances made in elucidating the mechanism of ICL repair with a focus on the role of TLS polymerases. We describe the emerging models for how these enzymes contribute to and are regulated in ICL repair, discuss the key open questions and examine the implications for this pathway in anti-cancer therapy.

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

Cited by 18 publications

References 60 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

Mutagenic Bypass of an Oxidized Abasic Lesion-Induced DNA Interstrand Cross-Link Analogue by Human Translesion Synthesis DNA Polymerases

Involvement of translesion synthesis DNA polymerases in DNA interstrand crosslink repair

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