Removal of VirD2 from a synthetic T-DNA complex of Agrobacterium by the host DNA repair protein TDP2

During plant genetic transformation, Agrobacterium transfers to its host cell a single-stranded segment of DNA (T-DNA), as a covalent complex with the virulence protein VirD2, which is ultimately integrated into the plant genome. The covalently bound VirD2 molecule is an obstacle to the ligation of the T-DNA 5’-end to host genomic DNA; therefore, VirD2 must be cleaved from the VirD2-T-DNA complex (T-complex) before the integration is completed. An interesting idea was raised that TDP2 (tyrosine phosphodiesterase 2), a protein known to resolve protein-DNA adducts, could represent a pathway for VirD2 removal from the T-complex; however, this function has not been proven directly. Here, we developed a simple technique to generate fluorescently labeled VirD2-ssDNA complexes in vitro and explored the DNA substrate requirements for the VirD2-mediated formation of these complexes, establishing a tractable experimental system for studying VirD2 removal from synthetic T-complexes. Using this system, we demonstrated that TDP2, from mammalian or plant origin, can specifically cleave VirD2 from these synthetic T-complexes; this activity was Mg 2+ -dependent and required a conserved C-terminal histidine residue of TDP2. Our results strongly support the hypothesis that plant TDP2 can remove VirD2 from the T-DNA before its integration into the plant genome.