Characterization and engineering of a DNA polymerase reveals a single amino-acid substitution in the fingers subdomain to increase strand-displacement activity of A-family prokaryotic DNA polymerases
Results - We have characterized the large fragment of a DNA polymerase I originating from the marine psychrophilic bacterium Psychrobacillus sp. The enzyme showed optimal polymerase activity at pH 8–9 and 25–110 mM NaCl/KCl. The polymerase was capable of performing polymerase as well as robust strand-displacement DNA synthesis at ambient temperatures (25–37 °C). Through molecular evolution and screening of thousand variants we have identified a single amino-acid exchange of Asp to Ala at position 422 which induced a 2.5-fold increase in strand-displacement activity of the enzyme.
Transferring the mutation of the conserved Asp residue to corresponding thermophilic homologues from Ureibacillus thermosphaericus and Geobacillus stearothermophilus also resulted in a significant increase in the strand-displacement activity of the enzymes.
Conclusions - Substituting Asp with Ala at positon 422 resulted in a significant increase in strand-displacement activity of three prokaryotic A-family DNA polymerases adapted to different environmental temperatures i.e. being psychrophilic and thermophilic of origin. This strongly indicates an important role for the 422 position and the O1-helix for strand-displacement activity of DNA polymerase I. The D422A variants generated here may be highly useful for isothermal nucleic acid amplification at a wide temperature scale.