A LOCUS INVOLVED IN THE REGULATION OF REPLICATION IN PLASMID PSC101

The origin of replication of plasmid pSC101 contains three directly re peated sequences RS1, RS2, and RS3 separated by 22bp from two palindro mic sequences, IR1 and IR2, which are partially homologous to the dire ct repeats. These inverted repeat (IR) sequences overlap the promoter of the repA gene which encodes a protein essential for plasmid replica tion. We have shown that RepA binds to the RS sites as a monomer and t o the IR sites as a dimer. The influence of the IR1 site, and of the D NA segment that separates it from RS3, on plasmid copy number control has been studied in detail. We show that the integrity of IR1 is essen tial for efficient replication and plasmid stability, the critical sit e extending to the left of IR1 proper. We also show that the presence of IR1 modifies profoundly the binding properties of purified RepA pro tein to a segment of DNA containing the RS sequences. IR1 is separated from its homologous site on RS3 by approximately four turns of the DN A helix. Replication is abolished if this distance is increased by hal f a turn of the helix but it is restored if the distance is increased by a whole turn. These results suggest a DNA looping interaction, in t he initiation of replication, between the RepA dimer that binds IR1 an d the RepA monomers that bind the RS sequences.