title: The role of DNA polymerase ζ in translesion synthesis across bulky DNA adducts and cross-links in human cells.
abstract: Translesion DNA synthesis (TLS) is a cellular defense mechanism against genotoxins. Defects or mutations in specialized DNA polymerases (Pols) involved in TLS are believed to result in hypersensitivity to various genotoxic stresses. Here, DNA polymerase ζ (Pol ζ)-deficient (KO: knockout) and Pol ζ catalytically dead (CD) human cells were established and their sensitivity towards cytotoxic activities of various genotoxins was examined. The CD cells were engineered by altering the DNA sequence encoding two amino acids essential for the catalytic activity of Pol ζ, i.e., D2781 and D2783, to alanines. Both Pol ζ KO and CD cells displayed a prolonged cell cycle and higher incidence of micronuclei formation than the wild-type (WT) cells in the absence of exogenous genotoxic treatments, and the order of abnormality was CD>KO>WT cells. Both KO and CD cells exhibited higher sensitivity towards the killing effects of benzo[a]pyrene diol epoxide, mitomycin C, potassium bromate, N-methyl-N'-nitro-N-nitrosoguanidine, and ultraviolet C irradiation than WT cells, and there were no differences between the sensitivities of KO and CD cells. Interestingly, neither KO nor CD cells were sensitive to the cytotoxic effects of hydrogen peroxide. Since KO and CD cells displayed similar sensitivities to the genotoxins, we employed only KO cells to further examine their sensitivity to other genotoxic agents. KO cells were more sensitive to the cytotoxicity of 4-nitroquinoline N-oxide, styrene oxide, cisplatin, methyl methanesulfonate, and ethyl methanesulfonate than WT cells. However, the KO cells displayed sensitivity camptothecin, etoposide, bleomycin, hydroxyurea, crotonealdehyde, and methylglyoxal in a manner similar to the WT cells. Our results suggest that Pol ζ plays an important role in the protection of human cells by carrying out TLS across bulky DNA adducts and cross-links, but has no or limited role in the protection against strand-breaks in DNA.
authors: 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: Tetsuya
authors: T
authors: Suzuki
authors: Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
authors: Petr
authors: P
authors: Grúz
authors: Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
authors: Masamitsu
authors: M
authors: Honma
authors: Graduate School of Nanobioscience, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan.
authors: Noritaka
authors: N
authors: Adachi
authors: Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
authors: Takehiko
authors: T
authors: Nohmi
mesh_terms: D018929:Cell Culture Techniques; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D018736:DNA Adducts / Q000096:biosynthesis* / Q000235:genetics; D004249:DNA Damage; D021881:DNA, Catalytic / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000502:physiology*; D055786:Gene Knockout Techniques; D006801:Humans; D048629:Micronuclei, Chromosome-Defective / Q000139:chemically induced / Q000706:statistics & numerical data; D009153:Mutagens / Q000633:toxicity
chemical_list: D018736:DNA Adducts; D021881:DNA, Catalytic; D004268:DNA-Binding Proteins; D009153:Mutagens; C425929:DNA polymerase zeta; D004259:DNA-Directed DNA Polymerase; C118285:REV3L protein, human
keywords: Catalytically dead mutant; DNA polymerase ζ; Knockout; Translesion DNA synthesis