IDENTIFICATION OF A DOMAIN OF ESCHERICHIA-COLI PRIMASE REQUIRED FOR FUNCTIONAL INTERACTION WITH THE DNAB HELICASE AT THE REPLICATION FORK

Primase plays a key role in governing the sequence of events required on the lagging strand during a cycle of Okazaki fragment synthesis. To begin to probe the protein-protein interactions necessary for primase function at the replication fork, we have used limited trypsinolysis to separate primase into two functional domains, an N-terminal domain of 49 kDa (p49) and a carboxyl-terminal domain of 16 kDa (p16). p49 re tained primase activity in replication assays that utilized bacterioph age M13 DNA carrying the bacteriophage G4 origin of DNA replication as the template, but was inactive during general priming or the conversi on of phi X174 single-stranded circular (ss(c))-DNA to the replicative form (RF) and could not support lagging strand DNA synthesis at repli cation forks reconstituted with the phi X-type primosomal proteins and the DNA polymerase m holoenzyme. On the other hand, p16 inhibited tho se replication reactions that included the replication fork helicase, DnaB (general priming, phi X174 ss(c) --> RF, and at the replication f ork), but had no effect on those that did not (M13Gori ss(c) --> RF). These results demonstrate that p49 defines a domain of primase require d for catalytic activity, that p16 defines a domain of primase require d for functional interaction with DnaB, and that it is a protein-prote in interaction with DnaB that attracts primase to the replication fork .