MUTATIONAL STUDIES OF HUMAN DNA POLYMERASE-ALPHA - IDENTIFICATION OF RESIDUES CRITICAL FOR DEOXYNUCLEOTIDE BINDING AND MISINSERTION FIDELITY OF DNA-SYNTHESIS

Conserved site-directed mutations were introduced into the second most conserved amino acid region, region II, of the human DNA polymerase a lpha catalytic subunit. These mutants were expressed in the baculoviru s system and purified to near homogeneity. The mutants had polymerase activity ranging from 4 to 60% compared with the wild type polymerase alpha. Steady-state kinetic analysis of mutants G841A, D860A, D860S, D 860N, Y865S, and Y865F demonstrated no significant difference in their K(m) values for primer-template compared with that of the wild type e nzyme. 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 mu tants showed that mutant Y865S but not Y865F had a greater than 10-fol d higher misinsertion efficiency than the wild type enzyme Mg2+-cataly zed reactions. However, with Mn2+ as the metal activator, Y865S and Y8 65F demonstrated a 2- and 9-fold higher misinsertion efficiency, respe ctively. These results indicate that Asp860 and Tyr865 in region II of human DNA polymerase alpha are involved in incoming dNTP substrate bi nding. Using three deoxynucleotide structural analogs as probes, we sh ow that the nucleotide base is the structural requirement for dNTP bin ding with Tyr865. Furthermore, abolishing the hydrophobic phenyl ring side chain of Tyr865 by replacing tyrosine with serine rendered the en zyme resistant to aphidicolin. Results of these studies strongly sugge st that the phenyl ring of Tyr865 directly interacts with the nucleoti de base moiety of the dNTP and plays a critical role in the misinserti on fidelity of DNA synthesis. Although mutation of Gly841 to Ala did n ot affect the binding of primer-template, it had a significant decreas e in k(cat), an increase in K(m) for dNTP, a striking decrease of proc essivity, and also resistance to aphidicolin. Thus, mutation of this r esidue, Gly841, which is highly conserved among the alpha-like DNA pol ymerases, appears to affect both catalysis and substrate deoxynucleoti de binding. This suggests that Gly841 is essential for the maintenance of the overall structure of the polymerase alpha catalytic site.