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 kcat/Km for dCTP insertion opposite G and N2-AnthG, with 2- to 3-fold decreases in DNA binding affinity, compared to wild-type, and further showed 5- to 190-fold decreases in kcat/Km for next-base extension from C paired with N2-AnthG. The A471V variant showed 2- to 4-fold decreases in kcat/Km for correct nucleotide insertion opposite and beyond G (or N2-AnthG) compared to 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 kcat/Km 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 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.
The Y-family DNA polymerase REV1 is involved in replicative bypass of damaged DNA and G-quadruplex (G4) DNA. In addition to a scaffolding role in the replicative bypass, REV1 acts in a catalytic role as a deoxycytidyl transferase opposite some replication stall sites, e.g. apurinic/apyrimidinic (AP) sites, N2-guanyl lesions, and G4 sites. We characterized the biochemical properties of 12 reported germline missense variants of human REV1, including the N373S variant associated with high risk of cervical cancer, using the recombinant REV1 (residues 330–833) proteins and DNA templates containing a G, AP site, N2-CH2(2-naphthyl)G (N2-NaphG), or G4. In steady-state kinetic analyses, the F427L, R434Q, M656V, D700N, R704Q, and P831L variants displayed 2- to 8-fold decreases in kcat/Km for dCTP insertion opposite all four templates, compared to that of wild-type, while the N373S, M407L, and N497S showed 2- to 3-fold increases with all four and the former three or two templates, respectively. The F427L, R434Q, M656V, and R704Q variants also had 2- to 3-fold lower binding affinities to DNA substrates containing G, an AP site, and/or N2-NaphG than wild-type. Distinctively, the N373S variant had a 3-fold higher binding affinity to G4 DNA than wild-type, as well as a 2-fold higher catalytic activity opposite the first tetrad G, suggesting a facilitating effect of this variation on replication of G4 DNA sequences in certain human papillomavirus genomes. Our results suggest that the catalytic function of REV1 is moderately or slightly altered by at least nine genetic variations, and the G4 DNA processing function of REV1 is slightly enhanced by the N373S variation, which might provide the possibility that certain germline missense REV1 variations affect the individual susceptibility to carcinogenesis by modifying the capability of REV1 for replicative bypass past DNA lesions and G4 motifs derived from chemical and viral carcinogens.
with lower values in average coordination distance geometry in the catalytic metal A-site. Crystal structures of R96G revealed the loss of three H-bonds of residues Gly-96 and Tyr-93 with an incoming dNTP, due to the lack of an arginine, as well as a destabilized Tyr-93 side chain secondary to the loss of a cationinteraction between both side chains. These results provide a mechanistic basis for alteration in pol catalytic function with coordinating metals and genetic variation.Genomic DNA is continuously attacked by a variety of endogenous and exogenous agents in cells, and the persistent unrepaired lesions can lead to genomic mutations and related diseases such as cancer. DNA polymerases (pols), 2 as well as DNA repair enzymes, are key enzymes for maintaining or altering genomic integrity against DNA lesions during various DNA transactions in organisms. The DNA replicative mechanisms linked to DNA damage and repair are believed to contribute to producing various mutational signatures in human cancer genomes (1). At least 17 different human DNA polymerases have been identified to date, which differ in their functions in DNA replication, repair, recombination, and damage tolerance (2, 3).Y-family DNA polymerases, including pols , , , and REV1, are specialized in replicating through DNA lesions, so-called translesion DNA synthesis (TLS). These polymerases have low fidelity with undamaged DNA templates but have spacious and solvent-accessible active sites to allow the accommodation and replicative bypass of bulky and distorted DNA lesions (4). Individual Y-family polymerases play error-free or error-prone roles in TLS, depending on DNA lesion types in cells (5). At bulky carcinogen-derived N 2 -G DNA lesions (e.g. benzo[a]pyrene-diol epoxide-G), both pol and REV1 catalyze error-free TLS, but both pols and catalyze error-prone TLS (6 -14). By contrast, at UV-induced cyclobutane thymine dimers (T-T), only pol (but not the other Y-family pols) can catalyze errorfree TLS (15,16). Therefore, the overall balance toward errorfree TLS with all working polymerases at various DNA lesions might be crucial in preventing mutations from numerous genotoxic agents. Recently, we reported that catalytic (either hypoactive or hyperactive) alterations are found in a considerable number of human germline non-synonymous variants of Y-family pols , , and REV1 (17-19), which might potentially influence on the overall TLS capacity in the affected individuals.pol is exceptionally error-prone in DNA synthesis among polymerases, particularly opposite template bases G and T, due to its uniquely restricted active-site and related non-WatsonCrick base pairing (20 -22). pol is able to catalyze nucleotide insertion opposite a variety of DNA lesions, including N 2 -and O 6 -alkyl and aralkyl G adducts, 8-oxo-7,8-dihydroG (8-oxoG),
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