SPECIFIC BINDING OF MOBA, A PLASMID-ENCODED PROTEIN INVOLVED IN THE INITIATION AND TERMINATION OF CONJUGAL DNA TRANSFER, TO SINGLE-STRANDEDORIT DNA

MobA protein, encoded by the broad host-range plasmid R1162, is requir ed for conjugal mobilization of this plasmid. The protein is an essent ial part of the relaxosome, and is also necessary for the termination of strand transfer. In vitro, MobA is a nuclease specific for one of t he two DNA strands of the origin of transfer (oriT). The protein can c leave this strand at the same site that is nicked in the relaxosome, a nd can also ligate the DNA. We show here that purified MobA protein fo rms a complex that is specific for this single oriT strand. The comple x is unusually stable, with a half-life of approximately 95 min, is no t disrupted by hybridization with the complementary strand, and reform s rapidly after boiling. Both the inverted repeat within oriT, and the eight bases between this repeat and the site cleaved by MobA, are req uired for binding by the protein. Mutations reducing base complementar ity between the arms of the inverted repeat also decrease binding. Thi s effect is partially suppressed by second-site mutations restoring co mplementarity. These results parallel the effects of these mutations o n termination. Footprinting experiments with Pl nuclease indicate that the DNA between the inverted repeat and the nick site is protected by MobA, but that pairing between the arms of the repeat, which occurs i n the absence of protein, is partially disrupted. Our results suggest that termination of strand transfer during conjugation involves tight binding of the MobA protein to the inverted repeat and adjacent oriT D NA. This complex positions the protein for ligation of the ends of the transferred strand, to reform a circular plasmid molecule.