1. Restoring 3'-5' exonuclease activity of thermophilic Geobacillus DNA polymerase I using site-directed mutagenesis in active site.
- Author
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Rastgoo N, Sadeghizadeh M, Bambaei B, and Hosseinkhani S
- Subjects
- Cloning, Molecular, DNA Polymerase I genetics, Enzyme Activation, Escherichia coli genetics, Geobacillus genetics, Geobacillus isolation & purification, Hot Springs microbiology, Iran, Recombinant Proteins metabolism, Structure-Activity Relationship, Catalytic Domain genetics, DNA Polymerase I metabolism, Exodeoxyribonucleases genetics, Exodeoxyribonucleases metabolism, Geobacillus enzymology, Mutagenesis, Site-Directed
- 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. more...
- Published
- 2009
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