Open AccessThree Human Pol ι Variants with Impaired Polymerase Activity Fail to Rescue H2O2 Sensitivity in POLI-Deficient Cells
Author(s) -
Mina Yeom,
Jin-Kyung Hong,
Jaekwon Kim,
F. Peter Guengerich,
JeongYun Choi
Publication year - 2020
Publication title -
chemical research in toxicology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.031
H-Index - 156
eISSN - 1520-5010
pISSN - 0893-228X
DOI - 10.1021/acs.chemrestox.0c00127
Subject(s) - base excision repair , dna polymerase , microbiology and biotechnology , polymerase , wild type , dna polymerase mu , biology , dna repair , dna damage , dna , biochemistry , gene , mutant , circular bacterial chromosome
Human Y-family DNA polymerase (pol) ι is involved in translesion DNA synthesis (TLS) and base excision repair (BER) of oxidative DNA damage. Genetic variations may alter the function of pol ι and affect cellular susceptibility to oxidative genotoxic agents, but their effects remain unclear. We investigated the impacts of 10 human missense germline variations on pol ι function by biochemical and cell-based assays. Both polymerase and deoxyribose phosphate (dRP) lyase activities were determined utilizing recombinant pol ι (residues 1-445) proteins. The K209Q, K228I, and Q386R variants showed 4- to 53-fold decreases in specificity constants ( k cat / K m ) for dCTP insertion opposite G and 8-oxo-7,8-dihydroguanine compared to the wild-type. The R126C and K345E variants showed wild-type-like polymerase activity, although these two variants (as well as the R209Q, K228I, and Q386R variants) showed greater than 6-fold decreases in dRP lyase activity compared to the wild-type. A CRISPR/Cas9-mediated POLI knockout conferred higher sensitivity to H 2 O 2 in human embryonic kidney (HEK293) cells. Exogenous expression of the full-length wild-type, R126C, and K345E variants fully rescued the H 2 O 2 sensitivity in POLI -deficient cells, while full-length R209Q, K228I, and Q386R variants did not rescue the sensitivity. Our results indicate that the R126C and K345E variants (having wild-type-like polymerase activity, albeit impaired in dRP lyase activity) could fully rescue the H 2 O 2 sensitivity in POLI -deficient cells, while the R209Q, K228I, and Q386R variants, all impaired in polymerase and dRP lyase activity, failed to rescue the sensitivity, indicating the relative importance of TLS-related polymerase function of pol ι rather than its BER-related dRP lyase function in protection from oxidative stress. The possibility exists that the hypoactive pol ι variants increase the individual susceptibility to oxidative genotoxic agents.