Biochemical Characterization of Eight Genetic Variants of Human DNA Polymerase κ Involved in Error-Free Bypass across Bulky N2-Guanyl DNA Adducts

DNA polymerase (pol) kappa, one of the Y-family polymerases, has been shown to function in error-free translesion DNA synthesis (TLS) opposite the bulky N-2-guanyl DNA lesions induced by many carcinogens such as polycyclic aromatic hydrocarbons. We analyzed the biochemical properties of eight reported human pol kappa variants positioned in the polymerase core domain, using the recombinant pol kappa (residues 1-526) protein and the DNA template containing an N-2-CH2(9-anthracenyl)G (N-2-AnthG). The truncation R219X was devoid of polymerase activity, and the E419G and Y432S variants showed much lower polymerase activity than wild-type pol kappa. In steady-state kinetic analyses, E419G and Y432S displayed 20- to 34-fold decreases in k(cat)/K-m for dCTP insertion opposite G and N-2-AnthG compared to that of wild-type pol kappa. The L21F, I39T, and D189G variants, as well as E419G and Y432S, displayed 6- to 22-fold decreases in k(cat)/K-m for next-base extension from C paired with N-2-AnthG, compared to that of wild-type pol kappa. The defective Y432S variant had 4- to 5-fold lower DNA-binding affinity than wild-type, while a slightly more efficient S423R variant possessed 2- to 3-fold higher DNA-binding affinity. These results suggest that R219X abolishes and the E419G, Y432S, L21F, I39T, and D189G variations substantially impair the TLS ability of pol kappa opposite bulky N-2-G lesions in the insertion step opposite the lesion and/or the subsequent extension step, raising the possibility that certain nonsynonymous pol kappa genetic variations translate into individual differences in susceptibility to genotoxic carcinogens.