Mechanism of cleavage and ligation by FLP recombinase: classification of mutations in FLP protein by in vitro complementation analysis.
The FLP recombinase of the 2 microns plasmid of Saccharomyces cerevisiae is a member of the integrase family of site-specific recombinases. Recombination catalyzed by members of this family proceeds via the ordered cleavage and religation of four strands of DNA. Although the amino acid sequences of integrase family members are quite different, each recombinase maintains an absolutely conserved tetrad of amino acids (R-191, H-305, R-308, Y-343; numbers are those of the FLP protein). This tetrad is presumed to reflect a common chemical mechanism for cleavage and ligation that has evolved among all family members. The tyrosine is the nucleophile that causes phosphodiester bond cleavage and covalently attaches to the 3'-PO4 terminus, whereas the other three residues have been implicated in ligation of strands. It has recently been shown that cleavage by FLP takes place in trans; that is, a FLP molecule binds adjacent to the site of cleavage but receives the nucleophilic tyrosine from a molecule of FLP that is bound to another FLP-binding element (J.-W. Chen, J. Lee, and M. Jayaram, Cell 69:647-658, 1992). These studies led us to examine whether the ligation step of the FLP reaction is performed by the FLP molecule bound adjacent to the cleavage site (ligation in cis). We have found that FLP promotes ligation in cis. Furthermore, using in vitro complementation analysis, we have classified several mutant FLP proteins into one of two groups: those proteins that are cleavage competent but ligation deficient (group I) and those that are ligation competent but cleavage defective (group II). This observation suggests that the active site of FLP is composed of several amino acid residues from each of two FLP molecules.
Pan
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Department of Molecular and Medical Genetics, University of Toronto, Ontario, Canada.
Luetke
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Sadowski
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D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004254:DNA Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005816:Genetic Complementation Test; D046911:Macromolecular Substances; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D013379:Substrate Specificity
D005656:Fungal Proteins; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase