ACTIVE-SITE MAPPING OF THE CATALYTIC MOUSE PRIMASE SUBUNIT BY ALANINESCANNING MUTAGENESIS

In the eukaryotic cell, DNA synthesis is initiated by DNA primase asso ciated with DNA polymerase alpha. The eukaryotic primase is composed o f two subunits, p49 and p58, where the p49 subunit contains the cataly tic active site, Mutagenesis of the cDNA for the p49 subunit was initi ated to demonstrate a functional correlation of conserved residues amo ng the eukaryotic primases and DNA polymerases, Fourteen invariant cha rged residues in the smaller catalytic mouse primase subunit, p49, wer e changed to alanine, These mutant proteins were expressed, purified, and enzymatically characterized for primer synthesis, Analyses of the mutant proteins indicate that residues 104-111 are most critical for p rimer synthesis and form part of the active site, Alanine substitution in residues Glu(105), Asp(109) and Asp(111) produced protein with no detectable activity in direct primase assays, indicating that these re sidues may form part of a conserved carboxylic triad also observed in the active sites of DNA polymerases and reverse transcriptases, Ah oth er mutant proteins showed a dramatic decrease in catalysis, while muta tion of two residues, Arg(162) and Arg(163), caused an increase in K-m (NTP). Analysis of these mutant proteins in specific assays designed t o separately investigate dinucleotide formation (initiation) and elong ation of primer indicates that these two activities utilize the same a ctive site within the p49 subunit, Finally, mutations in three active site codons produced protein with reduced affinity with the p58 subuni t, suggesting that p58 may interact directly with active site residues .