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. (C) 1996 Academic Press Limited