The Control Mechanism for Lagging Strand Polymerase Recycling during Bacteriophage T4 DNA Replication

Abstract

Given the polarity of DNA duplex, replication by the leading strand polymerase is continuous whereas that by the lagging strand polymerase is discontinuous proceeding through Okazaki fragments. Yet the respective polymerases act processively, implying that the recycling of the lagging strand polymerase is a controlled process. We demonstrate that the rate of the lagging strand polymerase relative to that of fork movement affects Okazaki fragment size and generates ssDNA gaps. We show by using a substrate with limited priming sites that Okazaki fragments can be shifted to shorter lengths by varying the rate of the primase. We find that clamp and clamp loader levels affect both primer utilization and Okazaki fragment size, possibly implicating clamp loading onto the RNA primer in the mechanism of lagging strand polymerase recycling. We formulate a signaling model capable of rationalizing the distribution of Okazaki fragments under various conditions for this and possibly other replisomes.