Molecular cloning of the replication terminus of the plasmid R6K

Analyses of the intermediates of DNA replication of the R6K plasmid derivatives, RSF1040, RJHC12 and RJHC26 demonstrate the transient accumulation of open circular DNA molecules with a discontinuity in either the plus or the minus strand of the DNA. The location of this discontinuity is nonrandom and is near the terminus of DNA replication. The discontinuity is not due to the activation of a relaxation complex since neither RJHC12 nor RJHC26 are relaxable. The replication terminus is functional in a clone containing approx. a 2000 bp sub-fragment of the HindIII-2 fragment of R6K. The replication terminus temporarily arrests the progression of replication fork of the unidirectionally replicating plasmid pBR313 at a region approx. 800 bp from HindIII site located nearest to the BamHI site of the vector. Subcloning experiments reveal that the upper limit of the replication termination sequence is 216 bp in length.