|
KMID : 0578319970070030419
|
|
Molecules and Cells
1997 Volume.7 No. 3 p.419 ~ p.424
|
|
|
Improvement of the 3´-5´ Exonuclease Activity of Taq DNA Polymerae by Protein Engineering in the Active Site
|
|
Park, Yonghyun
Choi, hyeja/Lee, Dae Sil/Kim, Youngsoo
|
|
|
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 catalyzed polymerase reactions. Taq DNA polymerase is classified into the poll family which is represented by E. coli DNA polymerase Ⅰ. The three dimensional structural alignment of 3´-5´ exonuclease domains from the poll 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.
|
|
|
KEYWORD
|
|
|
|
|
|
FullTexts / Linksout information
|
|
|
|
|
|
Listed journal information
|
|
  
|
|