Primase couples leading- and lagging-strand DNA synthesis from oriC.

Abstract

Coupling of leading- and lagging-strand DNA synthesis at replication forks formed at Escherichia coli oriC has been studied in vitro using a replication system reconstituted with purified proteins. At low concentrations of primase (8 nM), the major replication products were multigenome-length molecules, generated by a rolling circle-type mechanism, and unit-length molecules. Rolling circle DNA replication was inhibited at high concentrations of primase (80 nM) and the major replication products were half-unit-length leading strands and a distinct population of short Okazaki fragments. At low primase concentrations, an asymmetric mode of DNA synthesis occurred. Each strand was made independently and initiation could occur outside of oriC. At high primase concentrations, initiation occurred exclusively at oriC and two coupled replication forks proceeded bidirectionally around the plasmid. Presumably, at low concentrations of primase, DnaB (the replication fork helicase) unwound the plasmid DNA before replication forks could form, leading to initiation at sites other than oriC. On the other hand, high concentrations of primase resulted in successful capture of the helicase leading to the formation at oriC of coupled replication forks capable of coordinated leading- and lagging-strand synthesis.