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

Qun Dong, Teresa S.F. Wang

Research output: Contribution to journalArticlepeer-review

29 Scopus citations

Abstract

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 Km values for deoxynucleoside triphosphate but not kcat 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 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.

Original languageEnglish (US)
Pages (from-to)21563-21570
Number of pages8
JournalJournal of Biological Chemistry
Volume270
Issue number37
DOIs
StatePublished - Sep 15 1995

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of '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'. Together they form a unique fingerprint.

Cite this