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.