TUS PREVENTS OVERREPLICATION OF ORIC PLASMID DNA

Minichromosome plasmid DNA templates containing oriC and two Ter sites , oriented as they are on the Escherichia coli chromosome, have been u sed to study the role of Tus in termination of bidirectional replicati on. In replication reactions reconstituted with purified proteins wher e it could be demonstrated that each active template was replicating b idirectionally, Tus was required to prevent extensive overreplication. In the presence of Tus, both replication forks terminated DNA synthes is at one or the other Ter site in an apparent stepwise manner. First, the progress of one replication fork was arrested by a properly orien ted Tus-Ter complex. Then, either because of steric hindrance resultin g from the stalled replication machinery of the first fork or because of the formation of a branched DNA structure, the progression of the s econd opposing fork was halted at the same site on the DNA template. I n the absence of Tus, overreplication required DNA ligase and arose vi a a template strand-switching mechanism. Thus, the role of Tus in E. c oli is more likely to prevent overreplication rather than to ensure ac curate termination.