Mutational studies of human DNA polymerase alpha. Lysine 950 in the third most conserved region of alpha-like DNA polymerases is involved in binding the deoxynucleoside triphosphate.
The function of a lysine residue, Lys950, of human DNA polymerase alpha located in the third most conserved region and conserved in all of the alpha-like polymerases was analyzed by site-directed mutagenesis. Lys950 was mutagenized to Arg, Ala, or Asn. The mutant enzymes were expressed in insect cells infected with recombinant baculoviruses and purified to near homogeneity. The mutant enzymes had specific activities ranging from 8 to 22% of the wild type. All three Lys950 mutants utilized Mn2+ as metal activator more effectively than the wild type enzyme and showed an increase in Km values for deoxynucleoside triphosphate but not k(cat) values in reactions with either Mg2+ or Mn2+ as the metal activator. Although mutation of the Lys950 residue caused an increase in Km values for deoxynucleoside triphosphates, mutations of Lys950 to Arg, Ala, or Asn did not alter the mutant enzymes' misinsertion efficiency in reactions with Mg2+ as a metal activator as compared with that of the wild type, suggesting that the base of the incoming deoxynucleoside triphosphate is not the structural feature interacting with the Lys950 side chain. In reaction with Mn2+ as a metal activator, all three Lys950 mutants had an improved fidelity for deoxynucleotide misinsertion compared to wild type. Inhibition studies of the three Lys950 mutant derivatives with an inhibitor, structural analogs of deoxynucleoside triphosphate, and pyrophosphate suggest that the deoxyribose sugar and beta-,gamma-phosphate groups are not the structural feature recognized by the Lys950 side chain.(ABSTRACT TRUNCATED AT 250 WORDS)
Dong
Q
Q
Department of Pathology, Stanford University School of Medicine, California 94305, USA.
Wang
T S
TS
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D004257:DNA Polymerase II / Q000737:chemistry* / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008239:Lysine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera; D013329:Structure-Activity Relationship; D014162:Transfection; D014780:Viruses / Q000201:enzymology
D003854:Deoxyribonucleotides; D011994:Recombinant Proteins; D004257:DNA Polymerase II; D008239:Lysine