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.
McIlwraith
M J
MJ
Institute of Biomedical and Life Sciences, University of Glasgow, UK.
Boocock
M R
MR
Stark
W M
WM
D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism*; D007834:Lasers; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D013379:Substrate Specificity; D019895:Transposases
D003432:Cross-Linking Reagents; D004278:DNA, Superhelical; D004254:DNA Nucleotidyltransferases; D019895:Transposases