The p58 subunit of human DNA primase is important for primer initiation, elongation, and counting.
The p58 subunit of human DNA primase contains a region, M288-K344, that is homologous to part of the 8 kDa domain of DNA polymerase beta. Since regions of a protein that are highly conserved evolutionarily often play important catalytic functions, we examined the effects of mutating this region of the p58 subunit on primase activity. Deleting M288-L313 of the p58 subunit results in a protein that binds to the primase p49 subunit but cannot support primer synthesis on any template when assays only contain Mg(2+) as the divalent metal. Including Mn(2+), a metal that stimulates initiation of primer synthesis, in the assays now allows the enzyme to synthesize primers at a rate only moderately lower than that of the wild-type enzyme on templates consisting solely of deoxycytidylates. While the enzyme is active under these conditions, it has lost the ability to synthesize primers of defined length (i.e., count). Alanine scanning mutagenesis of charged residues in this region revealed three amino acids, R302, R306, and K314, that play important roles in both primer initiation and translocation. Conversion of these residues to alanine interfered with initiation and significantly decreased the processivity of primase. Together, these studies indicate that this "pol beta-like" region of p58 is important for three distinct aspects of primer synthesis:; initiation, translocation, and counting. The implications of these results with respect to the biological role of the p58 subunit and the mechanism of primer synthesis are discussed.
Zerbe
Laura K
LK
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA.
Kuchta
Robert D
RD
D000595:Amino Acid Sequence; D001483:Base Sequence; D017124:Conserved Sequence; D019951:DNA Polymerase beta / Q000737:chemistry; D019915:DNA Primase / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013698:Templates, Genetic
D021122:Protein Subunits; D011994:Recombinant Proteins; D019915:DNA Primase; D019951:DNA Polymerase beta