DNA cleavage in trans by the active site tyrosine during Flp recombination: switching protein partners before exchanging strands.
Each recombination event mediated by the Flp recombinase is the sum of four strand breakage and reunion reactions executed in two steps of two-strand exchanges. The reaction requires four Flp monomers. The key catalytic residue in Flp is Tyr-343. Arg-191, His-305, and Arg-308 appear to facilitate the cleavage and exchange steps of recombination. These four residues constitute the invariant tetrad of the Int family site-specific recombinases. Complementation tests between "step-arrest" mutants of Flp suggest that each Flp protomer harbors a "fractional active site." Hybrid "half site-recombinase" complexes reveal that efficient catalysis occurs when the Arg-His-Arg triad is present on one Flp monomer and the active site Tyr on a second monomer. Strand cleavage by an Flp monomer occurs virtually exclusively on the half site to which its partner protein is bound (cleavage in trans), and almost never on the half site to which it is bound (cleavage in cis). Trans-cleavage by Flp can provide a means for functionally exchanging Flp monomers between two DNA partners. Such a mechanism would be germane to recombination, since cleavage and rejoining in cis can only restore the parental substrate configuration and cannot yield recombinants.
Chen
J W
JW
Department of Microbiology, University of Texas, Austin 78712.
Lee
J
J
Jayaram
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism; D005816:Genetic Complementation Test; D046911:Macromolecular Substances; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013329:Structure-Activity Relationship; D014443:Tyrosine
D005656:Fungal Proteins; D046911:Macromolecular Substances; D014443:Tyrosine; D004247:DNA; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase