Localization of the start sites of lagging‐strand replication of rolling‐circle plasmids from Gram‐positive bacteria

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Abstract
Summary: 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 ONA 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.