6dic

Publication Abstract from PubMed

4,6-Diamino-5-formamidopyrimidine (Fapy*dG) is an abundant form of oxidative DNA damage that is mutagenic and contributes to the pathogenesis of human disease. When Fapy*dG is in its nucleotide triphosphate form, Fapy*dGTP, it is inefficiently cleansed from the nucleotide pool by the responsible enzyme in Escherichia coli MutT and its mammalian homolog MTH1. Therefore, under oxidative stress conditions, Fapy*dGTP could become a pro-mutagenic substrate for insertion into the genome by DNA polymerases. Here, we evaluated insertion kinetics and high-resolution ternary complex crystal structures of a configurationally stable Fapy*dGTP analog, beta-C-Fapy*dGTP, with DNA polymerase beta. The crystallographic snapshots and kinetic data indicate that binding of beta-C-Fapy*dGTP impedes enzyme closure, thus hindering insertion. The structures reveal that an active site residue, Asp276, positions beta-C-Fapy*dGTP so that it distorts the geometry of critical catalytic atoms. Removal of this guardian side chain permits enzyme closure and increases the efficiency of beta-C-Fapy*dG insertion opposite dC. These results highlight the stringent requirements necessary to achieve a closed DNA polymerase active site poised for efficient nucleotide incorporation and illustrate how DNA polymerase beta has evolved to hinder Fapy*dGTP insertion.

A guardian residue hinders insertion of a Fapy*dGTP analog by modulating the open-closed DNA polymerase transition.,Smith MR, Shock DD, Beard WA, Greenberg MM, Freudenthal BD, Wilson SH Nucleic Acids Res. 2019 Jan 16. pii: 5289696. doi: 10.1093/nar/gkz002. PMID:30649431^[5]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.