title: Mutagenesis of a highly conserved lysine 340 of the PRD1 DNA polymerase.
abstract: All known family B DNA polymerases contain a conserved region of amino acids, KX6-7YG, which appears to be correspond to the 'finger' alpha helix O of the Klenow fragment of E. coli DNA polymerase I, a family A DNA polymerase. Toward the goal of establishing the evolutionary relationship between the family A and B DNA polymerases, we have employed site-directed mutagenesis to access the functional role of the invariant amino acid lysine-340 of the PRD1 DNA polymerase. We have replaced the lysine-340 with three amino acids: histidine, asparagine and glutamic acid, respectively. Mutant DNA polymerases were overexpressed and purified to near homogeneity. Our results showed that the modification of the lysine-340 of the PRD1 DNA polymerase abolishes the polymerase activity without affecting the 3' to 5' exonuclease activity. These results support the proposal that the KX6-7YG motif of the family B DNA polymerases may be analogous to the KX7YG motif of the family A DNA polymerases, suggesting that two family DNA polymerases share a common ancestor.
authors.affiliation: Department of Microbiology and Immunology, College of Medicine, University of Arizona, Tucson 85724.
authors.forename: W
authors.initials: W
authors.lastname: Zhu
authors.forename: M C
authors.initials: MC
authors.lastname: Leavitt
authors.forename: G
authors.initials: G
authors.lastname: Jung
authors.forename: J
authors.initials: J
authors.lastname: Ito
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D003090:Coliphages / Q000201:enzymology*; D004247:DNA / Q000096:biosynthesis; D017931:DNA Primers / Q000737:chemistry; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D005092:Exonucleases / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
chemical_list: D017931:DNA Primers; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases; D008239:Lysine