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 Es cherichia coli replication fork and requires precise coordination of t he enzymatic activities required. We have shown previously that the pr imase is recruited anew from solution for each cycle of Okazaki fragme nt synthesis and that association of primase with the replication fork is via a protein-protein interaction with the helicase, DnaB, We desc ribe here mutant primases that have an altered interaction with DnaB a nd that direct the synthesis of Okazaki fragments of altered length co mpared to the wild-type. The mutant primases were deficient only in th eir ability to participate in replication reactions where their entry to the DNA was provided by the initial protein-protein interaction wit h DnaB. The primer synthesis capacity of these proteins remained unaff ected, as was their ability to interact with the DNA polymerase III ho loenzyme. Neither replication fork rate nor the efficiency of primer u tilization was affected at replication forks programmed by the mutant enzymes. Thus, the interaction between DnaG and DnaB at the replicatio n fork is the primary regulator of the cycle of Okazaki fragment synth esis.