MUTATIONAL STUDIES OF HUMAN DNA-POLYMERASE-ALPHA - LYSINE-950 IN THE 3RD MOST CONSERVED REGION OF ALPHA-LIKE DNA-POLYMERASES IS INVOLVED IN BINDING THE DEOXYNUCLEOSIDE TRIPHOSPHATE

The function of a lysine residue, Lys(950), 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. Lys(950) 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 Lys(950) mutants utilized Mn2+ as metal activator more effectively than the wild type enzyme and showed an increase in K-m values for deoxynucleoside triphosphate but not k(cat) values in reactions with either Mg2+ or Mn2+ as the metal activator. Although mutation of the Lys(950) residue caused an increase in K-m values for deoxynucleoside triphosphates, mutations of Lys(950) 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 Lys(950) Side chain. In reaction with Mn2+ as a metal activator, all three Lys(950) mutants had an improved fidelity for deoxynucleotide misinsertion compared to wild type. Inhibition studies of the three Lys(950) 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 Lys(950) side chain. Comparison of the mutant enzymes to the wild type enzyme for their affinities for dCTP alpha S versus deoxynucleoside triphosphate suggests that this highly conserved Lys(950) is involved in interacting either directly or indirectly with the oxygen moiety of the alpha-phosphate of the incoming deoxynucleoside triphosphate.