Biochemical characterization of eight genetic variants of human DNA polymerase κ involved in error-free bypass across bulky N(2)-guanyl DNA adducts.
DNA polymerase (pol) κ, 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 κ variants positioned in the polymerase core domain, using the recombinant pol κ (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 κ. 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 κ. 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 κ. 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 κ 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 κ genetic variations translate into individual differences in susceptibility to genotoxic carcinogens.
Division of Pharmacology, Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine , Suwon, Gyeonggi-do 440-746, Republic of Korea.
Insil
I
Song
Eun-Jin
EJ
Kim
In-Hyeok
IH
Kim
Eun-Mi
EM
Park
Kyung Eun
KE
Lee
Joo-Ho
JH
Shin
F Peter
FP
Guengerich
Jeong-Yun
JY
Choi
D018736:DNA Adducts / Q000737:chemistry / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014644:Genetic Variation; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000099270:Y-Family DNA Polymerases
D018736:DNA Adducts; D004259:DNA-Directed DNA Polymerase; D011994:Recombinant Proteins; C404302:POLK protein, human; D000099270:Y-Family DNA Polymerases