THE EXTREME C-TERMINUS OF PRIMASE IS REQUIRED FOR INTERACTION WITH DNAB AT THE REPLICATION FORK

We have shown previously that a protein-protein interaction between Dn aG 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 m utant p16 proteins that did not interact with DnaB. Various mutagenesi s protocols were used to localize this interaction domain to the extre me C terminus of the primase. A mutant primase missing only the C-term inal 16 amino acids was isolated and its activities examined. This mut ant enzyme was fully active as a primase, but was incapable of interac ting with DnaB. Thus, the mutant primase could not support DNA synthes is in either the general priming reaction or during phi X174 complemen tary strand DNA replication. Alanine cluster mutagenesis and deletion analysis in p16 allowed the further localization of the interaction do main to the extreme C-terminal 8 amino acids in primase.