fulltext.study @t Gmail

Restoring 3′–5′ exonuclease activity of thermophilic Geobacillus DNA polymerase I using site-directed mutagenesis in active site

Paper ID Volume ID Publish Year Pages File Format Full-Text
24404 43513 2009 8 PDF Available
Title
Restoring 3′–5′ exonuclease activity of thermophilic Geobacillus DNA polymerase I using site-directed mutagenesis in active site
Abstract

Three-dimensional structure and alignment analyses of 3′–5′ exonuclease domain of DNA polymerase I from thermophilic Geobacillus sp. MKK show that the key catalytic amino acids in 3′–5′ exonuclease domain are changed and the enzyme looses the activity. In order to render the activity, a catalytic module is constructed in the active site using site-directed mutagenesis. Seven mutant clones of the enzyme are generated containing: M1 (V319D, E325L), M2 (A376D), M3 (D425F), M4 (InsY446, K450D), M12 (V319D, E325L, A376D), M123 (V319D, E325L, A376D, D425F), and M1234 (V319D, E325L, A376D, D425F, InsY446, K450D). In addition, a chimera MkkEc polymerase is constructed by exchanging 3′–5′ exonuclease domain of the MKK polymerase (residues 301–466) with the same domain of homologous Escherichia coli polymerase (residues 327–519). For the first time, all essential amino acids for the 3′–5′ exonuclease activity are introduced in one mutant. As a result, among all mutants, only M1234 and MkkEc mutants show significant 3′–5′ exonuclease activity. Moreover, M1234 mutant was kept most of its polymerase activity while the activity of MkkEc mutants is decreased dramatically compared to the wild type enzyme.

Keywords
3′–5′ Exonuclease; Site-directed mutagenesis; Geobacillus sp. MKK; DNA polymerase
First Page Preview
Restoring 3′–5′ exonuclease activity of thermophilic Geobacillus DNA polymerase I using site-directed mutagenesis in active site
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 144, Issue 4, December 2009, Pages 245–252
Authors
, , , ,
Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us