title: Significant contribution of the 3'→5' exonuclease activity to the high fidelity of nucleotide incorporation catalyzed by human DNA polymerase ϵ.
abstract: Most eukaryotic DNA replication is performed by A- and B-family DNA polymerases which possess a faithful polymerase activity that preferentially incorporates correct over incorrect nucleotides. Additionally, many replicative polymerases have an efficient 3'→5' exonuclease activity that excises misincorporated nucleotides. Together, these activities contribute to overall low polymerase error frequency (one error per 10(6)-10(8) incorporations) and support faithful eukaryotic genome replication. Eukaryotic DNA polymerase ϵ (Polϵ) is one of three main replicative DNA polymerases for nuclear genomic replication and is responsible for leading strand synthesis. Here, we employed pre-steady-state kinetic methods and determined the overall fidelity of human Polϵ (hPolϵ) by measuring the individual contributions of its polymerase and 3'→5' exonuclease activities. The polymerase activity of hPolϵ has a high base substitution fidelity (10(-4)-10(-7)) resulting from large decreases in both nucleotide incorporation rate constants and ground-state binding affinities for incorrect relative to correct nucleotides. The 3'→5' exonuclease activity of hPolϵ further enhances polymerization fidelity by an unprecedented 3.5 × 10(2) to 1.2 × 10(4)-fold. The resulting overall fidelity of hPolϵ (10(-6)-10(-11)) justifies hPolϵ to be a primary enzyme to replicate human nuclear genome (0.1-1.0 error per round). Consistently, somatic mutations in hPolϵ, which decrease its exonuclease activity, are connected with mutator phenotypes and cancer formation.
authors.affiliation: Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA The Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Walter J
authors.initials: WJ
authors.lastname: Zahurancik
authors.affiliation: Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Seth J
authors.initials: SJ
authors.lastname: Klein
authors.affiliation: Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA The Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA suo.3@osu.edu.
authors.forename: Zucai
authors.initials: Z
authors.lastname: Suo
mesh_terms: D020137:Base Pair Mismatch; D055162:Biocatalysis; D004247:DNA / Q000096:biosynthesis* / Q000378:metabolism; D004257:DNA Polymerase II / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D006801:Humans; D009711:Nucleotides / Q000378:metabolism; D000075223:Poly-ADP-Ribose Binding Proteins; D013379:Substrate Specificity
chemical_list: D009711:Nucleotides; D000075223:Poly-ADP-Ribose Binding Proteins; D004247:DNA; D004257:DNA Polymerase II; C119525:POLE protein, human; D005090:Exodeoxyribonucleases