title: Kinetic mechanism of DNA polymerization catalyzed by human DNA polymerase ε.
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.
authors.affiliation: Department of Chemistry and Biochemistry, ‡The Ohio State Biochemistry Program, and §Department of Molecular Genetics, The Ohio State University , Columbus, Ohio 43210, United States.
authors.forename: Walter J
authors.initials: WJ
authors.lastname: Zahurancik
authors.forename: Seth J
authors.initials: SJ
authors.lastname: Klein
authors.forename: Zucai
authors.initials: Z
authors.lastname: Suo
mesh_terms: D020134:Catalytic Domain; D004247:DNA / Q000378:metabolism; D004257:DNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D005092:Exonucleases / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D000075223:Poly-ADP-Ribose Binding Proteins
chemical_list: D000075223:Poly-ADP-Ribose Binding Proteins; D004247:DNA; D004257:DNA Polymerase II; D004259:DNA-Directed DNA Polymerase; C119525:POLE protein, human; D005092:Exonucleases
references.citation: J Biol Chem. 2000 May 26;275(21):16316-22
references.pmid: 10748013
references.citation: Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2579-83
references.pmid: 8464864
references.citation: Nucleic Acids Res. 2008 Apr;36(6):1990-2001
references.pmid: 18276639
references.citation: Biochemistry. 2004 Feb 24;43(7):2116-25
references.pmid: 14967051
references.citation: J Biol Chem. 2001 Oct 12;276(41):38090-6
references.pmid: 11477093
references.citation: J Biol Chem. 1986 Sep 15;261(26):12310-6
references.pmid: 3745189
references.citation: Nucleic Acids Res. 2011 Mar;39(5):1763-73
references.pmid: 21036870
references.citation: Science. 2007 Jul 6;317(5834):127-30
references.pmid: 17615360
references.citation: Biochemistry. 2004 Feb 24;43(7):2106-15
references.pmid: 14967050
references.citation: J Biol Chem. 1987 Nov 25;262(33):16212-23
references.pmid: 3316214
references.citation: J Biol Chem. 1999 Dec 24;274(52):37060-9
references.pmid: 10601264
references.citation: DNA Res. 2000 Aug 31;7(4):243-51
references.pmid: 10997874
references.citation: Trends Cell Biol. 2008 Nov;18(11):521-7
references.pmid: 18824354
references.citation: Nucleic Acids Res. 2007;35(5):1569-77
references.pmid: 17289756
references.citation: Biochemistry. 2009 Aug 11;48(31):7502-11
references.pmid: 19456143
references.citation: Nature. 1994 May 19;369(6477):207-12
references.pmid: 7910375
references.citation: Methods Enzymol. 2009;467:601-626
references.pmid: 19897109
references.citation: Genes Cells. 2003 Nov;8(11):873-88
references.pmid: 14622139
references.citation: Nucleic Acids Res. 2007;35(19):6588-97
references.pmid: 17905813
references.citation: Biochemistry. 2004 Nov 16;43(45):14317-24
references.pmid: 15533035
references.citation: Biochemistry. 1991 May 21;30(20):4835-43
references.pmid: 1645180
references.citation: Mol Cell. 2008 Apr 25;30(2):137-44
references.pmid: 18439893
references.citation: J Biol Chem. 1990 Mar 5;265(7):4072-83
references.pmid: 2406268
references.citation: Biochemistry. 2001 Jul 31;40(30):9014-22
references.pmid: 11467964
references.citation: J Biol Chem. 1992 Dec 25;267(36):25988-97
references.pmid: 1281479
references.citation: Biochemistry. 1991 May 21;30(20):4844-54
references.pmid: 2036355
references.citation: Nature. 1987 Apr 2-8;326(6112):471-5
references.pmid: 2882422
references.citation: Biochemistry. 2010 Aug 31;49(34):7344-50
references.pmid: 20666462
references.citation: Biochemistry. 1992 Nov 17;31(45):10984-94
references.pmid: 1332748
references.citation: J Biol Chem. 2011 Aug 19;286(33):28963-28977
references.pmid: 21705323
references.citation: Nucleic Acids Res. 2007;35(9):3076-86
references.pmid: 17452367
references.citation: J Biol Chem. 2004 Apr 23;279(17):16895-8
references.pmid: 14988392
references.citation: J Biol Chem. 1993 Nov 25;268(33):24607-13
references.pmid: 7693703
references.citation: PLoS One. 2013 May 16;8(5):e63489
references.pmid: 23696828
references.citation: Biochemistry. 1991 Jan 15;30(2):511-25
references.pmid: 1846298