Resolution of synthetic chi structures by the FLP site-specific recombinase.
The FLP site-specific recombinase is encoded by the two micron circle, an endogenous plasmid of Saccharomyces cerevisiae. FLP-mediated recombination in vitro proceeds via a short-lived Holliday (chi) intermediate. We have made a synthetic chi structure containing two FLP recognition target (FRT) sequences in order to investigate resolution by purified FLP protein. We found that incubation of this model substrate with FLP generated two pairs of linear products in equal quantities. Thus, resolution was equally likely to occur in either direction. Alteration of FLP binding sites, so as to inhibit binding, affected the direction of resolution; cleavage was reduced adjacent to the altered binding site. The overall efficiency of resolution increased when one FLP binding site was mutated. In investigating the series of mutated chi structures we found that resolution requires only two intact FLP binding sites. However, the non-specific protein-DNA interaction of additional FLP molecules may also be required. Thus, resolution is more tolerant of the loss of FLP binding sites than is the complete recombination reaction.
Dixon
J E
JE
Department of Molecular and Medical Genetics, University of Toronto, Ontario, Canada.
Sadowski
P D
PD
D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D004254:DNA Nucleotidyltransferases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D010957:Plasmids; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase