Agrobacterium tumefaciens VirD2 polypeptide, in the presence of VirD1, catalyzes a site- and strand-specific nicking reaction at the T-DNA border sequences. VirD2 is found tightly attached to the 5' end of the nicked DNA. The protein-DNA complex is presumably formed via a tyrosine residue of VirD2 (F. Durrenberger, A. Crameri, B. Hohn, and Z. Koukolikova-Nicola, Proc. Natl. Acad. Sci. USA 86:9154-9158, 1989). A mutational approach was used to study whether a tyrosine residue(s) of VirD2 is required for its activity. By site- specific mutagenesis, a tyrosine (Y) residue at position 29, 68, 99, 119, 121, 160, or 195 of the octopine Ti plasmid pTiA6 VirD2 was altered to phenylalanine (F). The Y-29-F or Y-121-F mutation completely abolished nicking activity of VirD2 in vivo in Escherichia coli. Two other substitutions, Y-68-F and Y-160-F, drastically reduced VirD2 activity. A substitution at position 99, 119, or 195 had no effect on VirD2 activity. Additional mutagenesis experiments showed that at position 29, no other amino acid could substitute for tyrosine without destroying VirD2 activity. At position 121, only a tryptophan (W) residue could be substituted. This, however, yielded a mutant protein with significantly reduced VirD2 activity. The nicked DNA from strains bearing a Y-68-F, Y-99-F, Y-119-F, Y-160-F, Y- 195-F, or Y-121-W mutation in VirD2 was always found to contain a tightly linked protein.