Characterisation of a DNA polymerase highly mutated along the template binding interface.
Phage display establishes a link between a polypeptide and its corresponding gene. It has been much used for the isolation of proteins binding to chosen molecular targets. A second link was designed more recently between a phage-displayed enzyme and its reaction product. Affinity chromatography for the product then allows the isolation of catalytically active enzymes and of their genes. Using this strategy, a polymerase with 15 mutations was selected by directed evolution of Thermus aquaticus DNA polymerase I. The kinetic characterisation reported here highlights the variant's broad template specificity and classifies this enzyme as a thermostable DNA-dependent and RNA-dependent DNA-polymerase.
Vichier-Guerre
Sophie
S
Département de Biologie Structurale et Chimie, Institut Pasteur, 28 rue du Dr. Roux, Paris 15, France.
Jestin
Jean-Luc
JL
D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics*; D007700:Kinetics; D009154:Mutation / Q000235:genetics*; D013698:Templates, Genetic; D013824:Thermus / Q000201:enzymology*
D004259:DNA-Directed DNA Polymerase
J Mol Biol. 1999 Feb 19;286(2):617-33
9973575
Nucleic Acids Res. 1991 Aug 11;19(15):4133-7
1908075
Nature. 1998 Jan 15;391(6664):251-8
9440688
Res Microbiol. 2005 Dec;156(10):961-6
16293399
Res Microbiol. 2001 Mar;152(2):187-91
11316373
Proc Natl Acad Sci U S A. 1999 Aug 17;96(17):9497-502
10449721
Proc Natl Acad Sci U S A. 1998 Sep 1;95(18):10523-8
9724736
Science. 1985 Jun 14;228(4705):1315-7
4001944
Biochimie. 2008 Sep;90(9):1273-8
18466773
J Am Chem Soc. 2004 Feb 18;126(6):1748-54
14871106
Angew Chem Int Ed Engl. 2006 Sep 18;45(37):6133-7
16838276
Proc Natl Acad Sci U S A. 2001 Apr 10;98(8):4552-7
11274352
Biochimie. 2008 Nov-Dec;90(11-12):1796-9
18706966
J Biotechnol. 2004 Sep 30;113(1-3):85-103
15380650
Chembiochem. 2001 Apr 2;2(4):253-9
11828452
Nat Biotechnol. 1998 Oct;16(10):955-60
9788353
Angew Chem Int Ed Engl. 1999;38(8):1124-7
25138517
Protein Eng. 2001 Dec;14(12):1043-52
11809935
J Biol Chem. 1992 Aug 15;267(23):16007-10
1644788
Proc Natl Acad Sci U S A. 2002 May 14;99(10):6597-602
12011423
Mol Biotechnol. 2003 May;24(1):1-10
12721491
Protein Sci. 1995 Nov;4(11):2411-23
8563639
Proc Natl Acad Sci U S A. 2000 May 9;97(10):5095-100
10805772