Improvement of the 3'-5' exonuclease activity of Taq DNA polymerase by protein engineering in the active site

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Title
Improvement of the 3'-5' exonuclease activity of Taq DNA polymerase by protein engineering in the active site
Author(s)
Young Hyun Park; Hye Ja Choi; Dae Sil Lee; Young Soo Kim
Bibliographic Citation
Molecules and Cells, vol. 7, no. 3, pp. 419-424
Publication Year
1997
Abstract
Taq DNA polymerase from Thermus aquaticus has been shown to be very useful in the polymerase chain reaction method. Taq DNA polymerase has a domain at its amino terminus (residue 1 to 291) that has a 5′-3′ exonuclease activity, a 3′-5′ exonuclease domain in the middle (residue 292 to 423), and a domain at its C-terminus that catalyzes polymerase reactions. Taq DNA polymerase is classified into the polI family which is represented by E. coli DNA polymerase I. The three dimensional structural alignment of 3′-5′ exonuclease domains from the polI family, DNA polymerases leads us to understand why Taq DNA polymerase does not carry out proof-reading in the polymerase chain reaction. Three sequence motifs, called ExoI, II, and III must be present in order to carry out proof-reading by the 3′-5′ exonuclease reaction in DNA polymerization, but Taq DNA polymerase contains none of them. The key catalytic module in the 3′-5′ exonuclease is two metal ions chelated by active-site carboxylic amino acids. In order to render the 3′-5′ exonuclease activity in Taq DNA polymerase, a catalytic module was constructured in the active site by protein engineering. The mutant Taq DNA polymerase shows twice as much the 3′-5′ exonuclease activity as that of wild-type DNA polymerase.
ISSN
1016-8478
Publisher
South Korea
Type
Article
Appears in Collections:
1. Journal Articles > Journal Articles
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