An 18-base-pair sequence is sufficient for termination of rolling-circle replication of plasmid pT181

Abstract

pT181 and related plasmids of gram-positive bacteria replicate by a rolling-circle mechanism. The replication initiator protein of pT181, RepC, has origin-specific nicking-closing activities. Replication of the plasmid pT181 leading strand initiates by covalent extension of the RepC-generated nick, and the origin of replication contains signals for both initiation and termination of DNA replication. We have investigated the sequence requirements for the initiation and termination steps by using plasmids containing two pT181 origins. In vitro replication experiments showed that 18-and 24-bp synthetic oligonucleotides containing the RepC nick site were active in the termination of replication. However, initiation of replication required a larger region which also includes the RepC binding site. Plasmids containing the 18-and 24-bp region were also found to be nicked by the RepC protein. Our results demonstrate that sequence requirements for initiation and termination of pT181 replication overlap, but while the RepC binding site is required for initiation, it is dispensable for termination.

Sequence requirements for the termination of rolling‐circle replication of plasmid pT181

“…3). As shown previously (Zhao and Khan, 1996), these miniplasmids were maintained at much higher copy numbers than the vector pSA7650 plasmid both at 32ЊC and 43ЊC, since replication supported by RepC provided in trans dominates in the presence of a fully functional pT181 origin over the pE194 replicon contained in pSA7650 (Fig. 3).…”

“…The in vivo and in vitro replication termination activities of the various IRII mutants, along with their RepCdependent nicking activities are summarized in Table 1. Previous studies have shown that the IRII region of the pT181 origin by itself is sufficient for the termination of pT181 replication (Zhao and Khan, 1996). We tested the termination and initiation activities of IRII mutants to determine the role of its sequence and structure in these steps.…”

“…Previous studies have shown that nicking by RepC is required, but not sufficient, for the initiation of replication (Gennaro et al, 1989;Zhao and Khan, 1996). We wished to determine whether this is also the case for the termination step.…”

“…Previous studies have shown that the origin of replication contains sequences necessary for both the initiation and termination of RC replication (Dotto et al, 1984;Gros et al, 1987;Murray et al, 1989;Sozhamannan et al, 1990;Iordanescu and Projan, 1988;Zhao and Khan, 1996). While the sequences required for initiation and termination overlap, a larger region is required for the initiation step (Gros et al, 1987;Zhao and Khan, 1996). pT181 is a member of a family of staphylococcal plasmids that replicate by an RC mechanism (reviewed by Gruss and Ehrlich, 1989;del Solar et al, 1993;Khan, 1996).…”

“…Previous studies have shown that both IRII and IRIII regions are required for the initiation of pT181 replication (Gennaro et al, 1989;Zhao and Khan, 1996). Recent studies have also shown that IRII by itself is sufficient for termination of pT181 replication, although it is inactive in initiation (Zhao and Khan, 1996).…”