PRIMASE COUPLES LEADING-STRAND AND LAGGING-STRAND DNA-SYNTHESIS FROM ORIC

Coupling of leading- and lagging-strand DNA synthesis at replication f orks formed at Escherichia coli oriC has been studied in vitro using a replication system reconstituted with purified proteins. At low conce ntrations of primase (8 nM), the major replication products were multi genome-length molecules, generated by a rolling circle-type mechanism, and unit-length molecules Rolling circle DNA replication was inhibite d at high concentrations of primase (80 nM) and the major replication products were half-unit-length leading strands and a distinct populati on of short Okazaki fragments. At low primase concentrations, an asymm etric mode of DNA synthesis occurred. Each strand was made independent ly and initiation could occur outside of oriC. At high primase concent rations, initiation occurred exclusively at oriC and two coupled repli cation 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 conce ntrations of primase resulted in successful capture of the helicase le ading to the formation at oriC of coupled replication forks capable of coordinated leading- and lagging-strand synthesis.