Helicase action of dnaB protein during replication from the Escherichia coli chromosomal origin in vitro

Abstract

Initiation of bidirectional replication from the origin of the Escherichia coli chromosome (oriC) proceeds through stages in which the components of the two replication forks are assembled. From a complex containing proteins dnaA, dnaB, and dnaC bound at oriC, the dnaB helicase moves in both directions to unwind the duplex. In the absence of replication, this unwinding generates a bubble at oriC coated by single strand binding protein. Addition of gyrase allows unwinding to proceed extensively in both directions from oriC at 60 base pairs/s/fork at 37 degrees C. This rate is sharply dependent on temperature and also stimulated by both primase and DNA polymerase III holoenzyme, even in the absence of DNA synthesis. Primer and DNA synthesis are efficient when coupled to template unwinding. DNA synthesis proceeds bidirectionally from oriC at a rate limited by unwinding. With extensive unwinding preceding DNA synthesis, initiations are not limited to oriC.