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• W2070291889 endingPage "7049" @default.
• W2070291889 startingPage "7041" @default.
• W2070291889 abstract "Eukaryotes require highly accurate and processive DNA polymerases to ensure faithful and efficient replication of their genomes. DNA polymerase ε (Polε) has been shown to catalyze leading-strand DNA synthesis during replication in vivo, but little is known about the kinetic mechanism of polymerization catalyzed by this replicative enzyme. To elucidate this mechanism, we have generated a truncated, exonuclease-deficient mutant of the catalytic subunit of human Polε (Polε exo-) and carried out pre-steady-state kinetic analysis of this enzyme. Our results show that Polε exo-, as other DNA polymerases, follows an induced-fit mechanism when catalyzing correct nucleotide incorporation. Polε exo- binds DNA with a Kd(DNA) of 79 nM and dissociates from the E·DNA binary complex with a rate constant of 0.021 s(-1). Although Polε exo- binds a correct incoming nucleotide weakly with a Kd(dTTP) of 31 μM, it catalyzes correct nucleotide incorporation at a fast rate constant of 248 s(-1) at 20 °C. Both a large reaction amplitude difference (42%) between pulse-chase and pulse-quench assays and a small elemental effect (0.9) for correct dTTP incorporation suggest that a slow conformational change preceding the chemistry step limits the rate of correct nucleotide incorporation. In addition, our kinetic analysis shows that Polε exo- exhibits low processivity during polymerization. To catalyze leading-strand synthesis in vivo, Polε likely interacts with its three smaller subunits and additional replication factors in order to assemble a replication complex and significantly enhance its polymerization processivity." @default.
• W2070291889 created "2016-06-24" @default.
• W2070291889 creator A5003952728 @default.
• W2070291889 creator A5027160775 @default.
• W2070291889 creator A5055093260 @default.
• W2070291889 date "2013-09-26" @default.
• W2070291889 modified "2023-09-29" @default.
• W2070291889 title "Kinetic Mechanism of DNA Polymerization Catalyzed by Human DNA Polymerase ε" @default.
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• W2070291889 doi "https://doi.org/10.1021/bi400803v" @default.
• W2070291889 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3859840" @default.
• W2070291889 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/24020356" @default.
• W2070291889 hasPublicationYear "2013" @default.
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