The FLP protein contacts both major and minor grooves of its recognition target sequence.

The FLP protein of the 2 microns plasmid of Saccharomyces cerevisiae promotes conservative site-specific recombination between DNA sequences that contain the FLP recognition target (FRT). FLP binds to each of the three 13 base pair symmetry elements in the FRT site in a site-specific manner. We have probed both major and minor groove contacts of FLP using dimethyl sulphate, monoacetyl-4-hydroxyaminoquinoline 1-oxide and potassium permanganate and find that the protein displays extensive interactions with residues of both the major and minor grooves of 10 base pairs of each symmetry element. We find no evidence that the FRT site assumes a single-stranded conformation upon FLP binding.

Synaptic intermediates promoted by the FLP recombinase.

We have devised a novel assay to trap nucleoprotein synaptic intermediates of the FLP recombination reaction. DNase I footprinting analysis of these intermediates indicates that synapsis is mediated by protein-protein interactions between FLP molecules bound to each FLP recombination target (FRT) site. Under certain conditions we have observed a synaptic structure in which the FRT sites have come together in an aberrant arrangement. Although our analysis shows that homology between the core sequences of the sites is not a prerequisite for synapsis, the data suggest that homology between cores dictates the directionality of the reaction. Many of the intermediates contain a Holliday junction indicating that the FLP protein has catalysed strand exchanges between the FRT sites. The general scheme of the assay should prove useful to analyse nucleoprotein intermediates in other site-specific recombination systems, and to investigate protein-protein and protein-DNA interactions in intermediates important for DNA replication and transcription.

The mechanism of loading of the FLP recombinase onto its DNA target sequence.

The FLP recombinase interacts with its target sequence with the formation of three distinct DNA-protein complexes. The first complex leaves neither a DNase footprint nor is the DNA protected from methylation by dimethyl sulfate. We have found, however, that the FLP protein is bound predominantly to only one of the three 13 base-pair (bp) symmetry elements. This asymmetric loading of the FLP site seems to require the presence of an adjacent directly repeated 13 bp element. We speculate that this asymmetric filling of the target site may be accompanied by the unique order of cleavage and exchange of DNA strands.

Isolation of intermediates in the binding of the FLP recombinase of the yeast plasmid 2-micron circle to its target sequence.

We describe a method for isolating and characterizing intermediates in the binding of the FLP recombinase, encoded by the yeast plasmid 2-micron circle to its target sequence. On a wild-type substrate, three specific complexes are formed. Footprinting analysis of the gel-purified complexes shows that each complex is the result of a unique FLP-DNA association. On the basis of the behavior of various FLP target sequences in the gel-binding assay, we propose a model describing the steps that lead to the formation of a stable FLP-DNA complex.

FLP site-specific recombinase of yeast 2-micron plasmid. Topological features of the reaction.

The 2-micron plasmid of the yeast Saccharomyces cerevisiae encodes a site-specific recombinase (FLP) that promotes inversion across a unique site contained in each of the 599-base-pair inverted repeats of the plasmid. We have studied the topological changes generated in supercoiled substrates after exposure to the purified FLP protein in vitro. When a supercoiled substrate bearing two FLP target sequences in inverse orientation is treated with FLP, the products are multiply knotted structures that arise as a result of random entrapment of interdomainal supercoils. Likewise, a supercoiled substrate bearing two target sequences in direct orientation yields multiply interlocked catenanes as the product. Both types of substrate seem to be able to undergo repeated rounds of recombination that result in products of further complexity. The FLP protein also acts as a site-specific topoisomerase during the recombination reaction.

The FLP protein of the 2-micron plasmid of yeast. Purification of the protein from Escherichia coli cells expressing the cloned FLP gene.

Most laboratory strains of the yeast Saccharomyces cerevisiae contain many copies of an autonomously replicating plasmid called 2-micron circle DNA. This plasmid codes for a site-specific recombinase, the FLP protein which promotes recombination across two 599-base pair inverted repeats of the plasmid DNA. We have cloned the FLP gene under the control of a strong Escherichia coli promoter and have hyperproduced the protein in that organism. Cell-free extracts from this source promote highly efficient site-specific recombination in vitro and we have used this activity to purify the FLP protein substantially. The enzyme acts efficiently on circular and linear substrates and requires only monovalent or divalent cations for activity.

The FLP recombinase of the 2 micron circle DNA of yeast: interaction with its target sequences.

We have studied the interaction of purified FLP protein with restriction fragments from the substrate 2mu circle DNA of yeast. We find that FLP protects about 50 bp of DNA from nonspecific nuclease digestion. The protected site consists of two 13 bp inverted repeat sequences separated by an 8 bp spacer region. A third 13 bp element is also protected by binding of the FLP protein. We demonstrate that FLP introduces single- and double-strand breaks into the substrate DNA. This site-specific cleavage occurs at the margins of the spacer region, generating 8 bp 5' protruding ends with 5'-OH and 3'-protein-bound termini. Binding to mutant sites and half-sites demonstrates that the third symmetry element is not important for binding and cleavage by the FLP protein. The integrity of the core region is important for the cleavage activity of FLP.