Premature termination of DNA replication in plasmids carrying two inversely oriented ColE1 origins

In Escherichia coli plasmids carrying two inversely oriented ColE1 origins, DNA replication initiates at only one of the two potential origins. The ot her silent origin acts as a replication fork barrier. Whether this barrier is permanent or simply a pausing site remains unknown. Here, we used a repe ated primer extension assay to may in vivo, at the nucleotide level, the 5' end of the nascent strand where initiation and blockage of replication for ks occurs. Initiation occurred primarily at the previously defined origin, however, an alternative initiation site was detected 17 bp upstream. At the barrier, the lagging strand also terminated at the main initiation site. T herefore, the 5' end of the nascent strand at the barrier was identical to that generated during initiation. This observation strongly suggests that b lockage of the replication fork at the silent origin is not just a pausing site but permanent, and leads to a premature termination event. (C) 2000 Ac ademic Press.