COORDINATED LEADING-STRAND AND LAGGING-STRAND SYNTHESIS AT THE ESCHERICHIA-COLI DNA-REPLICATION FORK .5. PRIMASE ACTION REGULATES THE CYCLE OF OKAZAKI FRAGMENT SYNTHESIS

Replication forks formed during rolling-circle DNA synthesis supported by a tailed form II DNA substrate in the presence of the primosome, the single-stranded DNA binding protein, and the DNA polymerase III holoenzyme (Pol III HE) that had been reconstituted from the purified subunits, beta, tau, and the gamma.delta complex, at limiting (with respect to nucleotide incorporation) concentrations of the Pol III core (alpha, epsilon, and theta) produced aberrantly small Okazaki fragments, while the synthesis of the leading strand was unperturbed. These small Okazaki fragments were not arrayed in tandem along the lagging-strand DNA template, but were separated by large gaps. Similarly structured synthetic products were not manufactured by replication forks reconstituted with higher, saturating concentrations of the Pol III core. Replication forks producing these small fragments could respond, by modulating the size of the Okazaki fragments produced, to variations in the concentration of NTPs or the primase, conditions that affect the frequency of priming on the lagging strand, but not to variation in the concentration of dNTPs, conditions that affect the frequency of utilization of the primers. Significantly longer Okazaki fragments (> 7 kilobases) could be produced in the presence of a limiting amount of Pol Ill core at low concentrations of the primase. These observations indicated that the production of small Okazaki fragments was not a result of a debilitated lagging-strand Pol Ill core, but rather a function of the time available for nascent strand synthesis during the cycle of events that are required for the manufacture of an Okazaki fragment and that it was the association of primase with the replication fork that keyed this cycle.