title: Sensitivity of human cells expressing low-fidelity or weak-catalytic-activity variants of DNA polymerase ζ to genotoxic stresses.
abstract: Translesion DNA polymerases (TLS pols) play critical roles in defense mechanisms against genotoxic agents. The defects or mutations of TLS pols are predicted to result in hypersensitivity of cells to environmental mutagens. In this study, human cells expressing DNA polymerase ζ (Pol ζ) variants with low fidelity or weak catalytic activity have been established with Nalm-6-MSH+ cells and their sensitivity to mutagenicity and cytotoxicity of benzo[a]pyrene diol epoxide (BPDE) and ultraviolet-C light (UV-C) was examined. The low-fidelity mutants were engineered by knocking-in DNA sequences that direct changes of leucine 2618 to either phenylalanine (L2618F) or methionine (L2618M) of Pol ζ. The weak-catalytic-activity mutants were generated by knocking-in DNA sequences that direct changes of either tyrosine 2779 to phenylalanine (Y2779F) or aspartate 2781 to asparagine (D2781N). In addition, a +1 frameshift mutation, i.e., CCC to CCCC, was introduced in the coding region of the TK1 gene to measure the mutant frequencies. Doubling time and spontaneous TK mutant frequencies of the established cell lines were similar to those of the wild-type cells. The low-fidelity mutants displayed, however, higher sensitivity to the mutagenicity of BPDE and UV-C than the wild-type cells although their cytotoxic sensitivity was not changed. In contrast, the weak-catalytic-activity mutants were more sensitive to the cytotoxicity of BPDE and UV-C than the wild-type cells, and displayed much higher sensitivity to the clastogenicity of BPDE than the wild-type cells in an in vitro micronucleus assay. These results indicate that human Pol ζ is involved in TLS across DNA lesions induced by BPDE and UV-C and also that the TLS plays important roles in induction of mutations, clastogenicity and in cellular survival of the damaged human cells. Similarities and differences in in vivo roles of yeast and human Pol ζ in genome integrity are discussed.
authors.affiliation: Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan. Electronic address: suzukite@hiroshima-u.ac.jp.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
authors.forename: Petr
authors.initials: P
authors.lastname: Grúz
authors.affiliation: Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
authors.forename: Masamitsu
authors.initials: M
authors.lastname: Honma
authors.affiliation: Graduate School of Nanobioscience, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan.
authors.forename: Noritaka
authors.initials: N
authors.lastname: Adachi
authors.affiliation: Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
authors.forename: Takehiko
authors.initials: T
authors.lastname: Nohmi
mesh_terms: D019943:Amino Acid Substitution; D001402:B-Lymphocytes / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology / Q000528:radiation effects; D001483:Base Sequence; D001580:Benzopyrenes / Q000494:pharmacology; D045744:Cell Line, Tumor; D002470:Cell Survival; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism; D016368:Frameshift Mutation; D015870:Gene Expression; D006801:Humans; D015162:Micronucleus Tests; D016297:Mutagenesis, Site-Directed; D009153:Mutagens / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011836:Radiation Tolerance; D012441:Saccharomyces cerevisiae; D013937:Thymidine Kinase / Q000235:genetics* / Q000378:metabolism; D014466:Ultraviolet Rays
chemical_list: D001580:Benzopyrenes; D004268:DNA-Binding Proteins; D009153:Mutagens; D012333:RNA, Messenger; C072019:benzo(a)pyrene diolepoxide I; D004247:DNA; D013937:Thymidine Kinase; C092717:thymidine kinase 1; C425929:DNA polymerase zeta; D004259:DNA-Directed DNA Polymerase; C118285:REV3L protein, human
keywords: DNA polymerase ζ; Low-fidelity mutants; Nalm-6-MSH+/TK(+/−); Translesion DNA synthesis; Weak-catalytic-activity mutants