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 k_cat/K_m for 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 k_cat/K_m for next-base extension from C paired with N²-AnthG. The A471V variant showed 2- to 4-fold decreases in k_cat/K_m for 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 k_cat/K_m for 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.