The minimal duplex DNA sequence required for site-specific recombination promoted by the FLP protein of yeast<i>in vitro</i>

Proteau, Gerald; Sidenberg, Deborah Gayle; Sadowski, Paul D.

doi:10.1093/nar/14.12.4787

Cited by 28 publications

(6 citation statements)

References 27 publications

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“…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, (-]) (24); pBA116, ( --[) (4); and pBA114, (]<-) (5). (The arrows represent the 13-bp symmetry elements and their relative orientations.…”

Section: Methodsmentioning

confidence: 99%

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

Amin¹,

Roca²,

Luetke³

et al. 1991

Mol. Cell. Biol.

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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...

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Section: Methodsmentioning

confidence: 99%

“…The gels were dried and exposed to X-ray film. (24). We therefore adapted the half-site assay described by Landy and coworkers for the lambda Int recombination system (22,23).…”

Section: Methodsmentioning

confidence: 99%

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

Amin¹,

Roca²,

Luetke³

et al. 1991

Mol. Cell. Biol.

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“…Two of the three symmetry elements (a and b) are in inverted orientation surrounding an 8-bp core region. The third symmetry element (c), which is dispensable for recombination both in vivo and in vitro (Jayaram, 1985;Gronostajski and Sadowski, 1985;Proteau et al, 1986), is in direct orientation with symmetry element b.The FLP protein promotes efficient recombination in vitro, and the reaction has been studied extensively. After binding specifically to the symmetry elements, FLP induces an acute bend in the FRT site Sadowski, 1989, 1990), cleaves the top or bottom strands of the FRT site (see vertical arrows, Fig.…”

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confidence: 99%

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Cleavage-dependent Ligation by the FLP Recombinase

Zhu

Sadowski

1995

Journal of Biological Chemistry

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The FLP recombinase of the 2 M plasmid of Saccharomyces cerevisiae belongs to the integrase family of recombinases whose members have in common four absolutely conserved residues (Arg-191, His-305, Arg-308, and Tyr-343). We have studied the mutant protein FLP R308K in which the arginine residue at position 308 has been replaced by lysine. Although FLP R308K was previously reported to be defective in ligation of certain substrates (Pan, G., Luetke, K., and Sadowski, P. D., Mol. Cell. Biol. 13, 3167-3175, 1993b), we show in this work that the protein is able to ligate those substrates that it can cleave (cleavage-dependent ligation activity). FLP R308K is defective in in vitro recombination and in strand exchange. It is able to carry out strand exchange at one of the two cleavage sites of the FLP recognition target site (FRT site), but is defective in strand exchange at the other cleavage site. These results are consistent with a model in which wild-type FLP initiates recombination only at one of the two cleavage sites. FLP R308K may be defective in the initiation of recombination.Most strains of the yeast Saccharomyces cerevisiae harbor 50 -100 copies of an autonomously replicating plasmid, the 2 M circle (Broach and Hicks, 1980). This plasmid is 6318 bp 1 in length and contains two identical 599-bp inverted repeats that separate a small and large unique region. One of the open reading frames of the plasmid encodes a site-specific recombinase called "FLP" that promotes reciprocal recombination across the inverted repeats. The result is that the yeast contains approximately equal amounts of two isoforms, the A and B forms of the plasmid.The targets of the FLP protein are the two FLP recognition target sites (FRT) that are within the 599-bp inverted repeats. The FRT sites consist of three 13-bp symmetry elements to which the FLP protein binds in a site-specific manner (see Fig. 1; Andrews et al. (1985Andrews et al. ( , 1987). Two of the three symmetry elements (a and b) are in inverted orientation surrounding an 8-bp core region. The third symmetry element (c), which is dispensable for recombination both in vivo and in vitro (Jayaram, 1985;Gronostajski and Sadowski, 1985;Proteau et al., 1986), is in direct orientation with symmetry element b.The FLP protein promotes efficient recombination in vitro, and the reaction has been studied extensively. After binding specifically to the symmetry elements, FLP induces an acute bend in the FRT site Sadowski, 1989, 1990), cleaves the top or bottom strands of the FRT site (see vertical arrows, Fig. 1), and promotes the reciprocal exchange of a pair of strands to form a Holliday-like intermediate (Holliday, 1964;Meyer-Leon et al., 1988Jayaram et al., 1988) which is then resolved by a second pair of strand exchanges to form reciprocally recombinant molecules (Dixon and Sadowski, 1993, 1994).The FLP protein is a member of the integrase family of site-specific recombinases whose members have in common four absolutely conserved residues (arginine 191, histidine 305, arginine 308...

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“…It is well known that the third 13 bp symmetry element ('c', See Fig. 1a) is not needed for efficient recombination by Flp in vivo and in vitro [18,19]. The FRT site contains a near-perfect 13 bp palindrome, (the a and b symmetry elements, Figure 1a) which may cause unpredicted effects when the vector is used.…”

Section: Discussionmentioning

confidence: 99%

The Flp double cross system a simple efficient procedure for cloning DNA fragments

Sadowski

2003

BMC Biotechnol

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Background: While conventional cloning methods using restriction enzymes and polynucleotide ligase are adequate for most DNAs, fragments made by the polymerase chain reaction are difficult to clone because the amplifying DNA polymerase tends to add untemplated nucleotides to the 3'-termini of the amplified strands. Conservative site-specific recombinases offer an efficient alternative to conventional cloning methods.

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The minimal duplex DNA sequence required for site-specific recombination promoted by the FLP protein of yeastin vitro

Cited by 28 publications

References 27 publications

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

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

Cleavage-dependent Ligation by the FLP Recombinase

The Flp double cross system a simple efficient procedure for cloning DNA fragments

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