Isolation and Characterization of a Thermophilic DNA Polymerase from Geobacillus sp. MKK and Restoring its 3ʹ-5ʹ Exonuclease Activity

Abstract Molecular phylogenetic analysis of a novel thermophilic eubacterium isolated from an Iranian hot spring using 16S rDNA sequence showed that the new isolate belongs to genera Geobacillus. DNA pol I gene from this isolate was amplified, cloned, sequenced, and the three-dimensional (3D) structure of deduced amino acid sequence was predicted. Sequence analysis revealed the gene is 2,631 bp long, encodes a protein of 876 amino acids with a calculated molecular mass of 99 kDa, and belongs to family A DNA polymerases. Comparison of 3′–5′exonuclease domain of Klenow fragment (KF) with corresponding region of newly identified DNA pol I (MF), the large fragment of Bacillus stearothermophilus DNA pol I (BF) and Klentaq1, revealed not only deletions in three regions compared to KF, but that three of the four critical metal-binding residues in KF (Asp355, Glu357, Asp424, and Asp501) are altered in MF as well. Predicted 3D structure and sequence alignments between MF and BF showed that all critical residues in the polymerase active site are conserved. However, the key catalytic amino acids in 3ʹ-5ʹ exonuclease domain are changed and the enzyme has lost the relevant 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 3ʹ-5ʹ 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 3ʹ-5ʹ exonuclease activity are introduced in one mutant. As a result, among all mutants, only M1234 and MkkEc mutants show significant 3ʹ-5ʹ 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: Molecular phylogenetics, Thermostable DNA pol I, Cloning, 3D structure prediction, 3ʹ-5ʹ Exonuclease Activity, Site-directed mutagenesis.