Timely release of both replication forks from oriC requires modulation of origin topology

Initiation of DNA replication at oriC occurs bidirectionally both in vivo a nd in vitro. Although the proteins involved in establishing the replication forks are known, little is known about the events that ensure that initiat ion is bidirectional. We show here that in the absence of DNA gyrase, repli cation fork progression from oriC on a plasmid template in vitro is unidire ctional, although both replication forks have formed at the origin. There w as no bias in the release of one fork or the other, ruling out protein bloc kage of one fork as a possible reason for the asymmetric release. Timely re lease of both forks required the presence of either DNA gyrase or topoisome rase IV, suggesting that modulation of the topology of the origin region is the governing factor.