Localization of the start sites of lagging-strand replication of rolling-circle plasmids from gram-positive bacteria.

A number of small, multicopy plasmids from Gram-positive bacteria replicate by an asymmetric rolling-circle mechanism. Previous studies with several of these plasmids have identified a palindromic sequence, SSOA, that acts as the single-strand origin (SSO) for the replication of the lagging-strand DNA. Although not all the SSOA sequences share DNA sequence homology, they are structurally very similar. We have used an in vitro system to study the lagging-strand replication of several plasmids from Gram-positive bacteria using the SSOA sequences of pT181, pE194 and pSN2 as representative of three different groups of Staphylococcus aureus plasmids. In addition, we have investigated the lagging-strand replication of the pUB110 plasmid that contains an alternative single-strand origin, SSOU. Our results confirm that RNA polymerase is involved in lagging-strand synthesis from both SSOA and SSOU-type lagging-strand origins. Interestingly, while initiation of lagging-strand DNA synthesis of pUB110 occurred predominantly at a single position within SSOU, replication of pT181, pSN2 and pE194 plasmids initiated at multiple positions from SSOA.