Mutational studies of human DNA polymerase α. Identification of residues critical for deoxynucleotide binding and misinsertion fidelity of DNA synthesis

Q. Dong, W. C. Copeland, T. S.F. Wang

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69 Scopus citations


Conserved site-directed mutations were introduced into the second most conserved amino acid region, region II, of the human DNA polymerase α catalytic subunit. These mutants were expressed in the baculovirus system and purified to near homogeneity. The mutants had polymerase activity ranging from 4 to 60% compared with the wild type polymerase α. Steady-state kinetic analysis of mutants G841A, D860A, D860S, D860N, Y865S, and Y865F demonstrated no significant difference in their K(m) values for primer-template compared with that of the wild type enzyme. In contrast, mutants D860A, Y865S, and Y865F showed a 5-10-fold increase in the K(m) for deoxynucleotide triphosphate (dNTP) compared with the wild type enzyme. DNA synthetic fidelity studies of these mutants showed that mutant Y865S but not Y865F had a greater than 10-fold higher misinsertion efficiency than the wild type enzyme in Mg2+-catalyzed reactions. However, with Mn2+ as the metal activator, Y865S and Y865F demonstrated a 2- and 9-fold higher misinsertion efficiency, respectively. These results indicate that Asp860 and Tyr865 in region II of human DNA polymerase α are involved in incoming dNTP substrate binding. Using three deoxynucleotide structural analogs as probes, we show that the nucleotide base is the structural requirement for dNTP binding with Tyr865. Furthermore, abolishing the hydrophobic phenyl ring side chain of Tyr865 by replacing tyrosine with serine rendered the enzyme resistant to aphidicolin. Results of these studies strongly suggest that the phenyl ring of Tyr865 directly interacts with the nucleotide base moiety of the dNTP and plays a critical role in the misinsertion fidelity of DNA synthesis. Although mutation of Gly841 to Ala did not affect the binding of primer-template, it had a significant decrease in k(cat), an increase in K(m) for dNTP, a striking decrease of processivity, and also resistance to aphidicolin. Thus, mutation of this residue, Gly841, which is highly conserved among the α-like DNA polymerases, appears to affect both catalysis and substrate deoxynucleotide binding. This suggests that Gly841 is essential for the maintenance of the overall structure of the polymerase α catalytic site.

Original languageEnglish (US)
Pages (from-to)24163-24174
Number of pages12
JournalJournal of Biological Chemistry
Issue number32
StatePublished - 1993
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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