Termination of DNA replication in vitro at a sequence-specific replication terminus

Abstract

The replication terminus of the drug resistance factor R6K has been cloned into the plasmid vectors pBR313 and pBR322. When the exogenously added DNA is replicated in vitro using cell extracts prepared from Escherichia coli, the plasmid replication terminus temporarily arrests the progression of the unidirectionally moving replication fork at or near the cloned terminator sequence. When the relative location of the terminator sequence is changed with respect to the replication origin, the point of arrest of the replication fork shifts correspondingly to the new location of the terminator. Termination of replication takes place in vitro regardless of whether the cell extracts used in the in vitro reaction are prepared from E. coli with a resident terminus sequence containing plasmid. From these observations we conclude that the termination of replication in vitro is identical or very similar to that observed in vivo, membrane association is not necessary for the activity of the replication terminus and the terminus sequence does not code for a transacting factor necessary for termination of replication. Therefore, any transacting factor which may be needed for the termination of replication must be coded by the host chromosome.