Role of the double-strand origin cruciform in pT181 replication

pT181 is a small rolling-circle plasmid from Staphylococcus aureus whose in itiator protein, RepC, melts die plasmid's double-strand origin (DSO) and e xtrudes a cruciform involving IR II, a palindrome flanking the initiation n ick site. We have hypothesized that the cruciform is required for initiatio n, providing a single-stranded region for the assembly of the replisome (R. Jin et al., 1997, EMBO J. 16. 4456-4566). In this study, we have tested th e requirement for cruciform extrusion by disrupting the symmetry of the IR II palindrome or by increasing its length. The modified DSOs were tested fo r replication with RepC in trans. Rather surprisingly, disruption of the IR II symmetry had no detectable effect on replication or on competitivity of the modified DSO, though plasmids with IR H disrupted were less efficientl y relaxed than the wild type by RepC. However, in conjunction with IR II di sruption, modification of the tight RepC binding site IR III blocked replic ation. These results define two key elements of the pT181 initiation mechan ism-the IR II conformation and the RepC binding site (IR III)-and they indi cate that pT181 replication initiation is sufficiently robust to be able to compensate for significant modifications in the configuration of the DSO. (C) 2001 Academic Press.