The extreme C terminus of primase is required for interaction with DnaB at the replication fork

Abstract

We have shown previously that a protein-protein interaction between DnaG and DnaB is required to attract the primase to the replication fork. This interaction was mediated by the C-terminal 16-kDa domain (p16) of the primase. A screen was developed that allowed the detection of mutant p16 proteins that did not interact with DnaB. Various mutagenesis protocols were used to localize this interaction domain to the extreme C terminus of the primase. A mutant primase missing only the C-terminal 16 amino acids was isolated and its activities examined. This mutant enzyme was fully active as a primase, but was incapable of interacting with DnaB. Thus, the mutant primase could not support DNA synthesis in either the general priming reaction or during φX174 complementary strand DNA replication. Alanine cluster mutagenesis and deletion analysis in p16 allowed the further localization of the interaction domain to the extreme C-terminal 8 amino acids in primase.