LOCALIZATION OF THE START SITES OF LAGGING-STRAND REPLICATION OF ROLLING-CIRCLE PLASMIDS FROM GRAM-POSITIVE BACTERIA

A number of small, multicopy plasmids from Grampositive bacteria repli cate by an asymmetric rolling-circle mechanism. Previous studies with several of these plasmids have identified a palindromic sequence, sso( A), that acts as the single-strand origin (SSO) for the replication of the lagging-strand DNA. Although not all the sso(A), sequences share DNA sequence homology, they are structurally very similar. We have use d an in vitro system to study the lagging-strand replication of severa l plasmids from Gram-positive bacteria using the sso(A) sequences of p T181, pE194 and pSN2 as representative of three different groups of St aphylococcus aureus plasmids. In addition, we have investigated the la gging-strand replication of the pUB110 plasmid that contains an altern ative single-strand origin, sso(U). Our results confirm that RNA polym erase is involved in lagging-strand synthesis from both sso(A) and sso (U)-type lagging-strand origins. Interestingly, while initiation of la gging-strand DNA synthesis of pUB110 occurred predominantly at a singl e position within sso(U), replication of pT181, pSN2 and pE194 plasmid s initiated at multiple positions from sso(A).