Identification of the functional domains of the FLP recombinase. Separation of the nonspecific and specific DNA-binding, cleavage, and ligation domains.
The FLP recombinase of the 2-microns plasmid of Saccharomyces cerevisiae binds to its recognition target (FRT) site, induces a bend in the DNA, and promotes DNA cleavage and strand ligation. We have subjected this protein to limited proteolysis and have purified three polypeptides: P13 (13 kDa), P21 (21 kDa), and P32 (32 kDa). These peptides are derived from the following regions of FLP: P13, amino acids 2-123; P21, amino acids 148-346; P32, amino acids 124-423. In this report, we show that P13 binds to DNA nonspecifically and P32, like P21, binds to the FRT site specifically. A single molecule of P32 is able to induce a bend in the DNA of 55 degrees, similar to that induced by intact FLP (63 degrees). P13 enhances the binding of P21 or P32 to the FRT site. Both P32 and P21 can catalyze DNA ligation in combination with P13. P32 can cleave and covalently attach to the FRT site in combination with P13, whereas P21 cannot. These results suggest that FLP contains two DNA-binding domains. A nonspecific DNA-binding region is located in the NH2-terminal 123 amino acids, whereas the region that imparts specific DNA-binding resides in amino acids 148-346. Only the regions in P13 and P21 are needed for ligation activity, but those in P13 and P32 are required for cleavage.
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Department of Molecular and Medical Genetics, University of Toronto, Canada.
Sadowski
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D001483:Base Sequence; D001665:Binding Sites; D004254:DNA Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003850:Deoxyribonuclease I; D010450:Endopeptidases; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D003850:Deoxyribonuclease I; D010450:Endopeptidases