The experiments outlined in this proposal are directed toward an understanding of the DNA processing reactions that occur during conjugative transfer of the broad host-range plasmid R1162. A model is proposed in which the origin of transfer (oriT) consists of two domains, one required for initial nicking of the DNA prior to transfer, and the other for subsequent recircularization of the transferred but overlapping regions of a large open reading frame (ORFI). Mutations will be isolated in oriT to identify and map the different functional domains. Proteins fragments of ORFT that are involved in different processing events of oriT will be purified, and their biochemical activities tested in vitro. Protein-DNA relaxation complex will be characterized to identify the processing intermediate related to this structure. DNA synthesis occurs from within oriT in cell extracts and is dependent on the plasmid genes for transfer. The origin and direction of this synthesis will be determined. ORFI overlaps rep2, a gene required for plasmid DNA replication. The genetic relationship between rep2 and ORFI will be clarified, and the involvement of the rep2 region in DNA synthesis from oriT will be assessed. Mutations in oriT will also be tested to see whether nicking is required for synthesis. Another mobilization protein, MobI, complements the rep2 region of ORFI in vivo, and inhibits the processing of single-stranded oriT DNA. MobI will be tested for support of DNA synthesis from oriT, and the target of this protein will be identified. Finally, the R1162 DNA strands that are transferred during conjugation will be identified by capturing this DNA in infectious particles.