The Interaction between Helicase and Primase Sets the Replication Fork Clock

The synthesis of an Okazaki fragment occurs once every 1-2 s at the Escherichia coli replication fork and requires precise coordination of the enzymatic activities required. We have shown previously that the primase is recruited anew from solution for each cycle of Okazaki fragment synthesis and that association of primase with the replication fork is via a protein-protein interaction with the helicase, DnaB. We describe here mutant primases that have an altered interaction with DnaB and that direct the synthesis of Okazaki fragments of altered length compared to the wild-type. The mutant primases were deficient only in their ability to participate in replication reactions where their entry to the DNA was provided by the initial protein-protein interaction with DnaB. The primer synthesis capacity of these proteins remained unaffected, as was their ability to interact with the DNA polymerase III holoenzyme. Neither replication fork rate nor the efficiency of primer utilization was affected at replication forks programmed by the mutant enzymes. Thus, the interaction between DnaG and DnaB at the replication fork is the primary regulator of the cycle of Okazaki fragment synthesis. The synthesis of an Okazaki fragment occurs once every 1-2 s at the Escherichia coli replication fork and requires precise coordination of the enzymatic activities required. We have shown previously that the primase is recruited anew from solution for each cycle of Okazaki fragment synthesis and that association of primase with the replication fork is via a protein-protein interaction with the helicase, DnaB. We describe here mutant primases that have an altered interaction with DnaB and that direct the synthesis of Okazaki fragments of altered length compared to the wild-type. The mutant primases were deficient only in their ability to participate in replication reactions where their entry to the DNA was provided by the initial protein-protein interaction with DnaB. The primer synthesis capacity of these proteins remained unaffected, as was their ability to interact with the DNA polymerase III holoenzyme. Neither replication fork rate nor the efficiency of primer utilization was affected at replication forks programmed by the mutant enzymes. Thus, the interaction between DnaG and DnaB at the replication fork is the primary regulator of the cycle of Okazaki fragment synthesis.