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Characterization of Nine Cancer-Associated Variants in Human DNA Polymerase ?.

ABSTRACT: Specialized DNA damage-bypass Y-family DNA polymerases contribute to cancer prevention by providing cellular tolerance to DNA damage that can lead to mutations and contribute to cancer progression by increasing genomic instability. Y-family polymerases can also bypass DNA adducts caused by chemotherapy agents. One of the four human Y-family DNA polymerases, DNA polymerase (pol) ?, has been shown to be specific for bypass of minor groove adducts and inhibited by major groove adducts. In addition, mutations in the gene encoding pol ? are associated with different types of cancers as well as with chemotherapy responses. We characterized nine variants of pol ? whose identity was inferred from cancer-associated single nucleotide polymorphisms for polymerization activity on undamaged and damaged DNA, their abilities to extend from mismatched or damaged base pairs at primer termini, and overall stability and dynamics. We find that these pol ? variants generally fall into three categories: similar activity to wild-type (WT) pol ? (L21F, I39T, P169T, F192C, and E292K), more active than WT pol ? (S423R), and less active than pol ? (R219I, R298H, and Y432S). Of these, only pol ? variants R298H and Y432S had markedly reduced thermal stability. Molecular dynamics (MD) simulations with undamaged DNA revealed that the active variant F192C and more active variant S423R with either correct or incorrect incoming nucleotide mimic WT pol ? with the correct incoming nucleotide, whereas the less active variants R219I, R298H, and Y432S with the correct incoming nucleotide mimic WT pol ? with the incorrect incoming nucleotide. Thus, the observations from MD simulations suggest a possible explanation for the observed experimental results that pol ? adopts specific active and inactive conformations that depend on both the protein variant and the identity of the DNA adduct.

SUBMITTER: Antczak NM

PROVIDER: S-EPMC6130208 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Characterization of Nine Cancer-Associated Variants in Human DNA Polymerase κ.

Antczak Nicole M NM Walker Alice R AR Stern Hannah R HR Leddin Emmett M EM Palad Carl C Coulther Timothy A TA Swett Rebecca J RJ Cisneros G Andrés GA Beuning Penny J PJ

Chemical research in toxicology 20180730 8

Specialized DNA damage-bypass Y-family DNA polymerases contribute to cancer prevention by providing cellular tolerance to DNA damage that can lead to mutations and contribute to cancer progression by increasing genomic instability. Y-family polymerases can also bypass DNA adducts caused by chemotherapy agents. One of the four human Y-family DNA polymerases, DNA polymerase (pol) κ, has been shown to be specific for bypass of minor groove adducts and inhibited by major groove adducts. In addition, ...[more]

PMID: 30004685

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Project description:DNA polymerase (pol) ? is the most error-prone among the Y-family polymerases that participate in translesion synthesis (TLS). Pol ? can bypass various DNA lesions, e.g., N(2)-ethyl(Et)G, O(6)-methyl(Me)G, 8-oxo-7,8-dihydroguanine (8-oxoG), and an abasic site, though frequently with low fidelity. We assessed the biochemical effects of six reported genetic variations of human pol ? on its TLS properties, using the recombinant pol ? (residues 1-445) proteins and DNA templates containing a G, N(2)-EtG, O(6)-MeG, 8-oxoG, or abasic site. The ?1-25 variant, which is the N-terminal truncation of 25 residues resulting from an initiation codon variant (c.3G > A) and also is the formerly misassigned wild-type, exhibited considerably higher polymerase activity than wild-type with Mg(2+) (but not with Mn(2+)), coinciding with its steady-state kinetic data showing a ?10-fold increase in kcat/Km for nucleotide incorporation opposite templates (only with Mg(2+)). The R96G variant, which lacks a R96 residue known to interact with the incoming nucleotide, lost much of its polymerase activity, consistent with the kinetic data displaying 5- to 72-fold decreases in kcat/Km for nucleotide incorporation opposite templates either with Mg(2+) or Mn(2+), except for that opposite N(2)-EtG with Mn(2+) (showing a 9-fold increase for dCTP incorporation). The ?1-25 variant bound DNA 20- to 29-fold more tightly than wild-type (with Mg(2+)), but the R96G variant bound DNA 2-fold less tightly than wild-type. The DNA-binding affinity of wild-type, but not of the ?1-25 variant, was ?7-fold stronger with 0.15 mM Mn(2+) than with Mg(2+). The results indicate that the R96G variation severely impairs most of the Mg(2+)- and Mn(2+)-dependent TLS abilities of pol ?, whereas the ?1-25 variation selectively and substantially enhances the Mg(2+)-dependent TLS capability of pol ?, emphasizing the potential translational importance of these pol ? genetic variations, e.g., individual differences in TLS, mutation, and cancer susceptibility to genotoxic carcinogens.

Dataset Information

Characterization of Nine Cancer-Associated Variants in Human DNA Polymerase ?.

Publications

Characterization of Nine Cancer-Associated Variants in Human DNA Polymerase κ.

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