title: DNA polymerase minor groove interactions modulate mutagenic bypass of a templating 8-oxoguanine lesion.
abstract: A major base lesion resulting from oxidative stress is 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxoG) that has ambiguous coding potential. Error-free DNA synthesis involves 8-oxoG adopting an anti-conformation to base pair with cytosine whereas mutagenic bypass involves 8-oxoG adopting a syn-conformation to base pair with adenine. Left unrepaired the syn-8-oxoG/dAMP base pair results in a G-C to T-A transversion. During base excision repair of this mispair, DNA polymerase (pol) β is confronted with gap filling opposite 8-oxoG. To determine how pol β discriminates between anti- and syn-8-oxoG, we introduced a point mutation (R283K) to alter insertion specificity. Kinetic studies demonstrate that this substitution results in an increased fidelity opposite 8-oxoG. Structural studies with R283K pol β show that the binary DNA complex has 8-oxoG in equilibrium between anti- and syn-forms. Ternary complexes with incoming dCTP resemble the wild-type enzyme, with templating anti-8-oxoG base pairing with incoming cytosine. In contrast to wild-type pol β, the ternary complex of the R283K mutant with an incoming dATP-analogue and templating 8-oxoG resembles a G-A mismatched structure with 8-oxoG adopting an anti-conformation. These results demonstrate that the incoming nucleotide is unable to induce a syn-8-oxoG conformation without minor groove DNA polymerase interactions that influence templating (anti-/syn-equilibrium) of 8-oxoG while modulating fidelity.
authors.affiliation: Laboratory of Structural Biology, National Institute of Environmental Health Sciences, NIH, PO Box 12233, Research Triangle Park, NC 27709-2233, USA.
authors.forename: Bret D
authors.initials: BD
authors.lastname: Freudenthal
authors.forename: William A
authors.initials: WA
authors.lastname: Beard
authors.forename: Samuel H
authors.initials: SH
authors.lastname: Wilson
mesh_terms: D019943:Amino Acid Substitution; D004247:DNA / Q000737:chemistry*; D004249:DNA Damage; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry; D006801:Humans; D008958:Models, Molecular; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D013698:Templates, Genetic
chemical_list: C024829:8-hydroxyguanine; D006147:Guanine; D004247:DNA; D019951:DNA Polymerase beta