FORMATION OF A PUTATIVE RELAXATION INTERMEDIATE DURING T-DNA PROCESSING DIRECTED BY THE AGROBACTERIUM-TUMEFACIENS VIRD1,D2 ENDONUCLEASE

During the initial stages of crown gall tumorigenesis, the T-DNA regio n of the Agrobacterium tumefaciens Ti-plasmid is processed, resulting in the production of T-DNA molecules that are subsequently transferred to the plant cell. Processing of the T-DNA in the bacterium involves the nicking of T-DNA border sequences by an endonuclease encoded by th e virD locus, and the subsequent tight (possibly covalent) association of the VirD2 protein with the 5' end of the processed single-stranded or double-stranded T-DNA molecule. To investigate the interaction of the VirD1,D2 endonuclease with a right T-DNA border, a set of plasmids containing both the border and virD sequences on the same high-copy-n umber replicon has been constructed and introduced into Escherichia co li. In this model system a tight nucleoprotein complex is formed betwe en the relaxed double-stranded substrate plasmid and the VirD2 protein . This putative T-DNA processing complex may be analogous to the coval ent relaxation complex formed between the pilot protein and plasmid DN A during bacterial conjugation. VirD2 attachment to the relaxed substr ate plasmid was resistant to denaturing agents but sensitive to S1 nuc lease digestion, indicating a single-stranded region near the site of protein attachment. We speculate that this structure may be an interme diate formed prior to T-strand unwinding from the substrate plasmid in a host bacterium.