Origin and Fate of the 3′ Ends of Single-Stranded DNA Generated by Conjugal Transfer of Plasmid R1162

Abstract

ABSTRACT During conjugation, a single strand of DNA is cleaved at the origin of transfer ( oriT ) by the plasmid-encoded relaxase. This strand is then unwound from its complement and transferred in the 5′-to-3′ direction, with the 3′ end likely extended by rolling-circle replication. The resulting, newly synthesized oriT must then be cleaved as well, prior to recircularization of the strand in the recipient. Evidence is presented here that the R1162 relaxase contains only a single nucleophile capable of cleaving at oriT , with another molecule therefore required to cleave at a second site. An assay functionally isolating this second cleavage shows that this reaction can take place in the donor cell. As a result, there is a flux of strands with free 3′ ends into the recipient. These ends are susceptible to degradation by exonuclease I. The degree of susceptibility is affected by the presence of an uncleaved oriT within the strand. A model is presented where these internal oriT s bind and trap the relaxase molecule covalently bound to the 5′ end of the incoming strand. Such a mechanism would result in the preferential degradation of transferred DNA that had not been properly cleaved in the donor.