DNA recognition, strand selectivity, and cleavage mode during integrase family site-specific recombination.
We have probed the association of Flp recombinase with its DNA target using protein footprinting assays. The results are consistent with the domain organization of the Flp protein and with the general features of the protein-DNA interactions revealed by the crystal structures of the recombination intermediates formed by Cre, the Flp-related recombinase. The similarity in the organization of the Flp and Cre target sites and in their recognition by the respective recombinases implies that the overall DNA-protein geometry during strand cleavage in the two systems must also be similar. Within the functional recombinase dimer, it is the interaction between two recombinase monomers bound on either side of the strand exchange region (or spacer) that provides the allosteric activation of a single active site. Whereas Cre utilizes the cleavage nucleophile (the active site tyrosine) in cis, Flp utilizes it in trans (one monomer donating the tyrosine to its partner). By using synthetic Cre and Flp DNA substrates that are geometrically restricted in similar ways, we have mapped the positioning of the active and inactive tyrosine residues during cis and trans cleavage events. We find that, for a fixed substrate geometry, Flp and Cre cleave the labile phosphodiester bond at the same spacer end, not at opposite ends. Our results provide a model that accommodates local heterogeneities in peptide orientations in the two systems while preserving the global functional architecture of the reaction complex.
Tribble
G
G
Department of Microbiology, University of Texas, Austin, Texas 78712, Faculty of Applied Marine Sciences, Cheju University, Cheju City 690756, South Korea.
Ahn
Y T
YT
Lee
J
J
Dandekar
T
T
Jayaram
M
M
D000595:Amino Acid Sequence; D001419:Bacteria; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000235:genetics*; D016172:DNA Fingerprinting; D019426:Integrases / Q000235:genetics*; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae
D004247:DNA; D019426:Integrases