Biochemical characterization of eight genetic variants of human DNA polymerase ? involved in error-free bypass across bulky N(2)-guanyl DNA adducts.

ABSTRACT: DNA polymerase (pol) ?, 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 ? variants positioned in the polymerase core domain, using the recombinant pol ? (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 ?. In steady-state kinetic analyses, E419G and Y432S displayed 20- to 34-fold decreases in kcat/Km for dCTP insertion opposite G and N(2)-AnthG compared to that of wild-type pol ?. The L21F, I39T, and D189G variants, as well as E419G and Y432S, displayed 6- to 22-fold decreases in kcat/Km for next-base extension from C paired with N(2)-AnthG, compared to that of wild-type pol ?. 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 ? 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 ? genetic variations translate into individual differences in susceptibility to genotoxic carcinogens.

SUBMITTER: Song I 

PROVIDER: S-EPMC7002036 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature