Site-specific recombination by Tn3 resolvase, photocrosslinked to its supercoiled DNA substrate.

Site-specific recombination, transposition, and retroviral integration reactions involve the collaborative action of multiple identical protein subunits, making it difficult to determine the catalytic functions and fate of a subunit at any particular DNA binding site of the substrate. To investigate the mechanism of catalysis by the site-specific recombinase Tn3 resolvase, we fixed specific subunits to their binding sites by laser photocrosslinking, using a partially synthetic supercoiled DNA substrate containing photoreactive nucleotides. Crosslinked resolvase subunits were able to participate in a complete recombination reaction, demonstrating that the interaction of the subunit with its binding site persists throughout the reaction, and thus placing limitations on acceptable models for the catalytic mechanism.