Mutational studies of human DNA polymerase α: Lysine 950 in the third most conserved region of α-like DNA polymerases is involved in binding the deoxynucleoside triphosphate

The function of a lysine residue, Lys⁹⁵⁰, of human DNA polymerase α located in the third most conserved region and conserved in all of the α-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 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 Lys⁹⁵⁰ residue caused an increase in K_m 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 β-,γ-phosphate groups are not the structural feature recognized by the Lys⁹⁵⁰ side chain. Comparison of the mutant enzymes to the wild type enzyme for their affinities for dCTPαS versus deoxynucleoside triphosphate suggests that this highly conserved Lys⁹⁵⁰ is involved in interacting either directly or indirectly with the oxygen moiety of the α-phosphate of the incoming deoxynucleoside triphosphate.