Restoring 3'-5' exonuclease activity of thermophilic Geobacillus DNA polymerase I using site-directed mutagenesis in active site.
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.
Rastgoo
Nasrin
N
Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran 14115-175, Iran.
Sadeghizadeh
Majid
M
Bambaei
Bijan
B
Hosseinkhani
Saman
S
D020134:Catalytic Domain / Q000235:genetics*; D003001:Cloning, Molecular; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D056506:Geobacillus / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D045482:Hot Springs / Q000382:microbiology; D007492:Iran; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship
D011994:Recombinant Proteins; D004256:DNA Polymerase I; D005090:Exodeoxyribonucleases