Restoring 3'-5' exonuclease activity of thermophilic Geobacillus DNA polymerase I using site-directed mutagenesis in active site.
In: Journal of biotechnology, Jg. 144 (2009-12-01), Heft 4, S. 245-52
academicJournal
Zugriff:
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.
Titel: |
Restoring 3'-5' exonuclease activity of thermophilic Geobacillus DNA polymerase I using site-directed mutagenesis in active site.
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Autor/in / Beteiligte Person: | Rastgoo, N ; Sadeghizadeh, M ; Bambaei, B ; Hosseinkhani, S |
Zeitschrift: | Journal of biotechnology, Jg. 144 (2009-12-01), Heft 4, S. 245-52 |
Veröffentlichung: | Amsterdam : Elsevier Science Publishers, c1984-, 2009 |
Medientyp: | academicJournal |
ISSN: | 1873-4863 (electronic) |
DOI: | 10.1016/j.jbiotec.2009.09.006 |
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