LOCALIZED DENATURATION OF ORIT DNA WITHIN RELAXOSOMES OF THE BROAD-HOST-RANGE PLASMID R1162

The broad-host-range, multicopy plasmid R1162 is efficiently mobilized during conjugation by the self-transmissible plasmid R751. The relaxo some, a complex of plasmid DNA and R1162-encoded proteins, forms at th e origin of transfer (oriT) and is required for mobilization. Transfer is initiated by strand- and site-specific nicking of the DNA within t his structure. We show by probing with potassium permanganate that ori T DNA is locally melted within the relaxosome, in the region from the inverted repeat to the site that is nicked. Mutations in this region o f oriT, and in genes encoding the protein components of the relaxosome , affect both nicking and melting of the DNA. The nicking protein in t he relaxosome is MobA, which also ligates the transferred linear, sing le strand at the termination of a round of transfer. We propose that t here is an underlying similarity in the substrates for these two MobA- dependent, DNA-processing reactions. We also show that MobA has an add itional role in transfer, beyond the nicking and resealing of oriT DNA .