Synapsis, strand scission, and strand exchange induced by the FLP recombinase: analysis with half-FRT sites.

Abstract

We have used a previously described cross-linking assay and half-FRT site substrates to examine the requirements for synapsis, strand exchange, and strand scission. The cross-linking assay showed that the minimum functional FRT site needed for synapsis contains two inverted FLP-binding elements surrounding an 8-bp core. This indicates that four FLP molecules interact with four binding elements in a synaptic complex. The analysis using half-sites showed that the enzyme can catalyze efficient strand exchange between a half-site and the intact FRT site. The reaction occurred only if the half-site had at least 2 bp but no more than 4 bp of the adjoining core sequence. The exchange occurred exclusively at the regions of limited core homology between the respective half-site and the FRT site. Site-specific recombinases promote reciprocal exchanges between short, defined, homologous DNA sequences. The use of in vitro systems for several different site-specific recombinases has made it possible to examine the proteinprotein and protein-DNA interactions involved in the many transition steps of the reactions (9,18,26). This has led to the formulation of models that describe the mechanisms of the various reactions (10,11).The FLP protein encoded by the 2,um plasmid of yeast cells is one such site-specific recombinase whose in vitro reaction is amenable to molecular dissection (6,8,29). The target sequence of the FLP protein, the FRT site, consists of three 13-bp symmetry elements designated a, b, and c (Fig. 1A). Two of these elements are in inverted orientation and separated by an 8-bp core sequence. The recombinase catalyzes cleavage of the phosphodiester bond at the junction of the symmetry elements and the core region (4, 7). This cleavage is accompanied by the formation of a 3' phosphotyrosyl linkage (12). These two junctions are the points of strand exchange between two synapsed FRT sites (15,21). When the protein is bound to the inverted symmetry elements, a sharp bend occurs within the core sequence (27,28).We recently developed a method of isolating synaptic and postsynaptic intermediates of the FLP recombination reaction (1). An analysis of these intermediates indicated that synapsis occurred by protein-protein interactions between FLP molecules bound to symmetry elements a and b of each of the paired wild-type FRT sites. In addition, we showed that synapsis occurred in the absence of core homology, and we argued that core homology was important only after synapsis to allow the progression of the reaction via a Holliday intermediate.We have now further investigated the requirement for * Corresponding author.synapsis by using the assay mentioned above. In addition, we have used half-FRT sites to determine the requirements for strand cleavage and strand exchange. MATERIALS AND METHODSConstruction of plasmids. Plasmids were used as vectors as well as sources of partial FRT sites. The nomenclature of plasmids and the respective FRT sites they contained is as follows: pGP25, (-] Figure 1B summarizes the various cons...

Active-site assembly and mode of DNA cleavage by Flp recombinase during full-site recombination.

“…Half-site substrates (Fig. 1B), originally designed for the A Int reaction (15) and subsequently adapted for the Flp reaction (2,21,22), have simplified the mechanistic analysis of site-specific recombination. A half site contains one Flp-binding element, one scissile phosphodiester, and one 5'-hydroxyl group that can act as a phosphoryl acceptor.…”

Cleavage-dependent Ligation by the FLP Recombinase

“…After a 60-min incubation, the reactions were stopped by adding sample buffer to achieve final concentrations of 10% glycerol, 3% SDS, 60 mM Tris-Cl (pH 6.8), and 5% ␤-mercaptoethanol. The samples were then boiled for 5 min and analyzed on a 15% SDS-polyacrylamide gel (Laemmli, 1970;Amin et al, 1991;Pan et al, 1993a).…”

“…In Vitro Recombination Assay-The assay was done essentially as described by Amin et al (1991) and Pan et al (1993b). FLP catalyzes recombination between a plasmid pLB112-generated FRT site (Beatty and Sadowski, 1988) and a synthetic FRT site (FS15, Table II), producing two recombinant molecules with different sizes which can be analyzed on an 8% denaturing gel.…”

DNA-promoted assembly of the active tetramer of the Mu transposase.

“…A similar type of complex assembly between individual monomers bound to isolated single sites is seen for the FLP site-specific recombinase of the yeast 2Ix plasmid. On DNA sites that carry only half of the normal binding site dyad, binding of one FLP monomer recruits another to make dimer and some trimer and tetramer complexes (Qian et al 1990;Amin et al 1991). Formation of the dimer by protein-protein contacts, brings two DNA sites together and forms a complex active in recombination.…”