DETERMINATION OF SEQUENCE SPECIFICITY BETWEEN A PLASMID REPLICATION INITIATOR PROTEIN AND THE ORIGIN OF REPLICATION

Staphylococcal plasmids of the pT181 family replicate by a rolling cir cle mechanism, requiring the activities of a plasmid-specified Rep pro tein. The initiation event involves site-specific phosphodiester bond cleavage by Rep within the replication origin, ori. In vitro the Rep p roteins also display type-I topoisomerase activity specific for this p lasmid family Although the single site of bond cleavage, ICR II, is co nserved among all members of the pT181 family, the plasmid-specific Re p proteins are able to discriminate between family members in vivo, in itiating replication only from the cognate origin. The basis of such s pecificity is believed to be due to a non-covalent binding interaction between Rep and a DNA sequence adjacent to the site of phosphodiester bond cleavage. Using the RepD protein specified by plasmid pC221, we present data for the physical parameters of RepD:oriD complex formatio n. Quantification of the relative strengths of the non-covalent intera ctions for different but related ori target sequences, measured by gel mobility shift experiments, has yielded data that are in accord with the known specificity of the protein in vivo. Oligonucleotide competit ion experiments demonstrate that this interaction is indeed attributab le to the specificity determinant, ICR III. Protein-DNA crosslinking m ethods show that a carboxyl-terminal proteolytic fragment of RepD make s a specific interaction with the ICR III region of its cognate replic ation origin. Analysis of topoisomerase rates indicates that the inter action between ICR III and the carboxyl terminus of the protein is req uired before a productive interaction, namely the phosphodiester bond cleavage at the ICR II, can occur. (C) 1995 Academic Press Limited