FIDELITY STUDIES OF THE HUMAN DNA POLYMERASE-ALPHA - THE MOST CONSERVED REGION AMONG ALPHA-LIKE DNA-POLYMERASES IS RESPONSIBLE FOR METAL-INDUCED INFIDELITY IN DNA-SYNTHESIS

Mutational studies in the highly conserved region I domain of the huma n DNA polymerase alpha enzyme demonstrated a change in metal cation-sp ecific catalysis. Here, we extend the investigation to include the fid elity of DNA synthesis by these mutants, studying misinsertion, mispai r extension, and the nucleotide analog utilization. The fidelity of re gion I mutants and wild type human DNA polymerase alpha enzyme were an alyzed with either Mg2+ or Mn2+ as the metal activator. Despite the kn own mutagenic effect of Mn2+ in causing polymerases to misinsert nucle otides and to utilize dideoxynucleotides, we have found that two regio n I mutants, D1002N and T1003S, which utilize Mn2+ in catalysis more e ffectively than Mg2+, actually have a 70- and 40-fold higher misinsert ion fidelity, respectively, in Mn2+-catalyzed reactions than that of t he wild type enzyme. The enhanced misinsertion fidelity of these two m utants in Mn2+-catalyzed reactions is due to K(m) discrimination of th e incorrect nucleotide where the D1002N and T1003S had a 850- and 62-f old higher K(m) for insertion of incorrect than correct nucleotide, re spectively. In Mg2+-catalyzed reactions, all of the region I mutants e xhibited similar misinsertion efficiencies as the wild type polymerase . Study of mispair extension showed that in Mn2+-catalyzed reactions, the wild type polymerase alpha enzyme readily extended mispair termini . In contrast, the two region I mutants, D1002N and T1003S, were unabl e to extend the mispaired termini in either Mg2+- or Mn2+-catalyzed re actions. These results suggest that the side chains of region I amino acids play an essential role in the Mn2+-induced infidelity during DNA synthesis by human DNA polymerase alpha. The effects of the metal act ivator on the utilization of two nucleotide analogs, 3'-azido-3'-deoxy thymidine triphosphate and ddCTP, by the region I mutants were also in vestigated.