Six Germline Genetic Variations Impair the Translesion Synthesis Activity of Human DNA Polymerase κ.
DNA polymerase (pol) κ efficiently catalyzes error-free translesion DNA synthesis (TLS) opposite bulky N2-guanyl lesions induced by carcinogens such as polycyclic aromatic hydrocarbons. We investigated the biochemical effects of nine human nonsynonymous germline POLK variations on the TLS properties of pol κ, utilizing recombinant pol κ (residues 1-526) enzymes and DNA templates containing an N2-CH2(9-anthracenyl)G (N2-AnthG), 8-oxo-7,8-dihydroguanine (8-oxoG), O6-methyl(Me)G, or an abasic site. In steady-state kinetic analyses, the R246X, R298H, T473A, and R512W variants displayed 7- to 18-fold decreases in kcat/Kmfor dCTP insertion opposite G and N2-AnthG, with 2- to 3-fold decreases in DNA binding affinity, compared to that of the wild-type, and further showed 5- to 190-fold decreases in kcat/Kmfor next-base extension from C paired with N2-AnthG. The A471V variant showed 2- to 4-fold decreases in kcat/Kmfor correct nucleotide insertion opposite and beyond G (or N2-AnthG) compared to that of the wild-type. These five hypoactive variants also showed similar patterns of attenuation of TLS activity opposite 8-oxoG, O6-MeG, and abasic lesions. By contrast, the T44M variant exhibited 7- to 11-fold decreases in kcat/Kmfor dCTP insertion opposite N2-AnthG and O6-MeG (as well as for dATP insertion opposite an abasic site) but not opposite both G and 8-oxoG, nor beyond N2-AnthG, compared to that of the wild-type. These results suggest that the R246X, R298H, T473A, R512W, and A471V variants cause a general catalytic impairment of pol κ opposite G and all four lesions, whereas the T44M variant induces opposite lesion-dependent catalytic impairment, i.e., only opposite O6-MeG, abasic, and bulky N2-G lesions but not opposite G and 8-oxoG, in pol κ, which might indicate that these hypoactive pol κ variants are genetic factors in modifying individual susceptibility to genotoxic carcinogens in certain subsets of populations.
Division of Pharmacology, Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine , Suwon, Gyeonggi-do 16419, Republic of Korea.
Jae-Kwon
JK
Kim
Division of Pharmacology, Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine , Suwon, Gyeonggi-do 16419, Republic of Korea.
Mina
M
Yeom
Division of Pharmacology, Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine , Suwon, Gyeonggi-do 16419, Republic of Korea.
Jin-Kyung
JK
Hong
Division of Pharmacology, Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine , Suwon, Gyeonggi-do 16419, Republic of Korea.
Insil
I
Song
Department of New Biology, Daegu Gyeongbuk Institute of Science and Technology , Daegu 42988, Republic of Korea.
Young-Sam
YS
Lee
Department of Biochemistry, Vanderbilt University School of Medicine , Nashville, Tennessee 37232-0146, United States.
F Peter
FP
Guengerich
Division of Pharmacology, Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine , Suwon, Gyeonggi-do 16419, Republic of Korea.
Jeong-Yun
JY
Choi
D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism*; D014644:Genetic Variation / Q000235:genetics*; D006801:Humans; D008958:Models, Molecular; D008968:Molecular Conformation; D000099270:Y-Family DNA Polymerases
D004259:DNA-Directed DNA Polymerase; C404302:POLK protein, human; D000099270:Y-Family DNA Polymerases