Homology requirements for ligation and strand exchange by the FLP recombinase.
The FLP recombinase of the 2-microns plasmid of Saccharomyces cerevisiae belongs to the integrase family whose members form a covalent bond between a conserved tyrosine of the recombinase and the 3'-phosphoryl group at the site of cleavage. Ligation takes place when the 5'-OH generated during the cleavage step attacks the phosphotyrosine bond and reforms a phosphodiester bond. When the incoming 5'-OH is from the partner duplex, strand exchange occurs. The FLP recognition target (FRT) contains two inverted 13-base pair (bp) FLP binding sequences that surround an 8-bp core region. It has been shown that heterology in the core regions of the recombinase FLP recognition target sites can dramatically impair recombination. Therefore, it was of interest to study the homology requirements of the core sequence for FLP-mediated ligation. Using nicked duplex substrates containing mismatches in the core sequence, we have demonstrated that the FLP ligation reaction can tolerate mismatches at all positions in the 8-bp core except the position immediately adjacent to the cleavage site. Using half-FRT substrates that contain a single-stranded core sequence, we showed that 4 base pairs adjacent to the cleavage site in the core are required for FLP to execute ligation with a single-stranded oligonucleotide. FLP is also able to ligate the protruding single strand on a half-FRT site to the opposite strand to form a hairpin. We have studied the effect of the base composition of the protruding 8-nucleotide single strand upon the efficiency of hairpin ligation. These studies revealed the importance of intrastrand complementarity in the formation of hairpin by FLP. Hence we conclude that the homology in the position adjacent to the cleavage site is most important, and the degree of the homology required is dependent on the nature of the ligation assay.
Zhu
X D
XD
Department of Molecular and Medical Genetics, University of Toronto, Ontario, Canada.
Pan
G
G
Luetke
K
K
Sadowski
P D
PD
D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000378:metabolism; D019000:Phosphotyrosine; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014443:Tyrosine / Q000031:analogs & derivatives
D009838:Oligodeoxyribonucleotides; D019000:Phosphotyrosine; D014443:Tyrosine; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase