title: DNA polymerase proofreading: active site switching catalyzed by the bacteriophage T4 DNA polymerase.
abstract: DNA polymerases achieve high-fidelity DNA replication in part by checking the accuracy of each nucleotide that is incorporated and, if a mistake is made, the incorrect nucleotide is removed before further primer extension takes place. In order to proofread, the primer-end must be separated from the template strand and transferred from the polymerase to the exonuclease active center where the excision reaction takes place; then the trimmed primer-end is returned to the polymerase active center. Thus, proofreading requires polymerase-to-exonuclease and exonuclease-to-polymerase active site switching. We have used a fluorescence assay that uses differences in the fluorescence intensity of 2-aminopurine (2AP) to measure the rates of active site switching for the bacteriophage T4 DNA polymerase. There are three findings: (i) the rate of return of the trimmed primer-end from the exonuclease to the polymerase active center is rapid, >500 s(-1); (ii) T4 DNA polymerase can remove two incorrect nucleotides under single turnover conditions, which includes presumed exonuclease-to-polymerase and polymerase-to-exonuclease active site switching steps and (iii) proofreading reactions that initiate in the polymerase active center are not intrinsically processive.
authors: Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada.
authors: Elizabeth
authors: E
authors: Fidalgo da Silva
authors: Linda J
authors: LJ
authors: Reha-Krantz
mesh_terms: D015075:2-Aminopurine / Q000737:chemistry; D019943:Amino Acid Substitution; D020137:Base Pair Mismatch; D001665:Binding Sites; D002384:Catalysis; D004247:DNA / Q000096:biosynthesis / Q000737:chemistry; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D005453:Fluorescence; D007700:Kinetics; D009711:Nucleotides / Q000378:metabolism; D013941:Thymine / Q000737:chemistry; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D009711:Nucleotides; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; D015075:2-Aminopurine; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; D013941:Thymine
references: Cell. 1999 Oct 15;99(2):155-66
references: 10535734
references: J Biol Chem. 1983 Aug 25;258(16):9649-56
references: 6604051
references: Genetics. 2001 May;158(1):177-86
references: 11333228
references: Cell. 2001 Jun 1;105(5):657-67
references: 11389835
references: Biochemistry. 2002 Apr 2;41(13):4399-406
references: 11914087
references: J Biol Chem. 2002 Oct 25;277(43):40640-9
references: 12189135
references: Proc Natl Acad Sci U S A. 2002 Nov 26;99(24):15560-5
references: 12429860
references: EMBO J. 2004 Apr 7;23(7):1494-505
references: 15057282
references: EMBO J. 2004 Apr 7;23(7):1483-93
references: 15057283
references: Proc Natl Acad Sci U S A. 2004 Jun 1;101(22):8289-94
references: 15148377
references: Proc Natl Acad Sci U S A. 2004 Jun 1;101(22):8255-6
references: 15159531
references: Cold Spring Harb Symp Quant Biol. 1966;31:693-7
references: 5237208
references: Nature. 1969 Mar 22;221(5186):1128-32
references: 4975273
references: J Biol Chem. 1972 Jan 10;247(1):241-8
references: 4336040
references: J Biol Chem. 1972 Nov 25;247(22):7116-22
references: 4565077
references: J Mol Biol. 1977 Oct 15;116(1):115-23
references: 592380
references: J Biol Chem. 1982 Oct 25;257(20):12435-43
references: 6981648
references: Proc Natl Acad Sci U S A. 1983 Mar;80(6):1669-73
references: 6300866
references: Cell. 1988 Jun 17;53(6):837-40
references: 2838173
references: FASEB J. 1989 Jan;3(1):14-21
references: 2642867
references: J Biol Chem. 1992 Jul 15;267(20):14157-66
references: 1629215
references: Biochemistry. 1992 Nov 17;31(45):10984-94
references: 1332748
references: J Virol. 1993 Jan;67(1):60-6
references: 8380094
references: Nucleic Acids Res. 1993 Feb 25;21(4):787-802
references: 8451181
references: Crit Rev Biochem Mol Biol. 1993;28(2):83-126
references: 8485987
references: J Biol Chem. 1994 Feb 25;269(8):5635-43
references: 8119900
references: Biochemistry. 1994 Jun 21;33(24):7576-86
references: 8011623
references: Biochemistry. 1995 Jul 18;34(28):9185-92
references: 7619819
references: J Mol Biol. 1995 Nov 17;254(1):15-28
references: 7473755
references: J Biol Chem. 1996 Nov 15;271(46):28903-11
references: 8910538
references: Proc Natl Acad Sci U S A. 1997 Sep 30;94(20):10506-11
references: 9380669
references: Proc Natl Acad Sci U S A. 1998 Mar 31;95(7):3507-12
references: 9520396
references: J Biol Chem. 1998 Sep 4;273(36):22969-76
references: 9722519
references: J Biol Chem. 1999 Aug 27;274(35):25151-8
references: 10455197
references: Biochemistry. 2005 Dec 6;44(48):15674-84
references: 16313170
references: Curr Biol. 2006 Jan 24;16(2):202-7
references: 16431373
references: Biochemistry. 2006 Mar 7;45(9):2836-44
references: 16503638
references: J Biol Chem. 2007 Jan 12;282(2):1432-44
references: 17098747
references: Biochemistry. 2007 Jun 5;46(22):6559-69
references: 17497891
references: Mol Cell Biol. 2000 Oct;20(20):7490-504
references: 11003646