MUTAGENESIS OF A HIGHLY CONSERVED LYSINE-340 OF THE PRD1 DNA-POLYMERASE

Al known family B DNA polymerases contain a conserved region of amino acids, KX6-nG, which appears to be correspond to the 'finger' alpha he lix O of the Klenow fragment of E. coli DNA polymerase I, a family A D NA polymerase. Toward the goal of establishing the evolutionary relati onship between the family A and B DNA polymerases, we have employed si te-directed mutagenesis to access the functional role of the invariant amino acid lysine-340 of the PRD1 DNA polymerase. We have replaced th e lysine-340 with three amino acids: histidine, asparagine and glutami c acid, respectively. Mutant DNA polymerases were overexpressed and pu rified to near homogeneity. Our results showed that the modification o f the lysine-340 of the PRD1 DNA polymerase abolishes the polymerase a ctivity without affecting the 3' to 5' exonuclease activity. These res ults support the proposal that the KX6-7YG motif of the family B DNA p olymerases may be analogous to the KX7YG motif of the family A DNA pol ymerases, suggesting that two family DNA polymerases share a common an cestor.