The virD operon of Agrobacterium tumefaciens Ti plasmid encodes a DNA-relaxing enzyme

Abstract

The virD locus of Agrobacterium tumefaciens Ti plasmid encodes functions necessary for endonucleolytic cleavage of transferred DNA (T-DNA) prior to its transfer to plant cells. For the overproduction of the VIRD proteins in Escherichia coli a tac-virD operon fusion was constructed. A significant increase in the accumulation of VIRD proteins was observed in a lon protease-deficient E. coli host. The presence of an overlapping open reading frame (ORF) upstream of the VIRD1 coding sequence had a negative effect on VIRD1 production. A preparation containing VIRD proteins catalyzes the conversion of supercoiled (form I) DNA to relaxed (form IV) DNA. This activity is similar to that of a DNA topoisomerase. The relaxation activity lacks DNA sequence specificity, requires magnesium ion, and has no requirement for an energy source. Studies with plasmids that had lost defined DNA segments encompassing various virD coding regions showed that VIRD1 is the DNA-relaxing enzyme. In a density gradient centrifugation experiment, the DNA-relaxing activity sedimented as a 21-kDa polypeptide. Earlier studies of Jayaswal et al. [Jayaswal, R., Veluthambi, K., Gelvin, S. & Slightom, J. (1987) (J. Bacteriol. 169, 5035-5045] have shown that in E. coli VIRD2 alone is not sufficient for endonucleolytic cleavage of T-DNA and requires VIRD1 for its activity.