FLP Recombinase of the 2 μm circle plasmid of Saccharomyces cerevisiae bends its DNA target

Schwartz, Carol; Sadowski, Paul D.

doi:10.1016/0022-2836(89)90310-0

Cited by 50 publications

(28 citation statements)

References 35 publications

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“…The Ty3 integration machinery could interact directly with proteins of the transcription complex or could recognize an altered DNA conformation induced by transcription factor binding. DNA bending is important for bacteriophage X (Goodman and Nash 1989;Snyder et al 1989) and the Saccharomyces FLP (Schwartz and Sadowski 1989) recombination reactions. Both transcription factors TFIIIC and TFIIIB induce a bend centered near the transcription initiation site when bound to a tRNA*~^^" gene (Leveillard et al 1991).…”

Section: Ty3 Targets Pol Ill-ttanscribed Genesmentioning

confidence: 99%

Ty3 integrates within the region of RNA polymerase III transcription initiation.

Chalker¹,

Sandmeyer²

1992

Genes Dev.

202

187

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Over 190 independent insertions into target plasmids of the retro virus-like element Ty3 were recovered and mapped. Ty3 was shown to insert upstream of tRNA, 5S, and U6 genes, all of which are transcribed by RNA polymerase III. Integration sites were within 1-4 nucleotides of the position of transcription initiation, even for one mutant gene where the polymerase III initiation site was shifted to a completely new context. Mutagenesis of a SUP2 tRNA gene target showed that integration required functional promoter elements but that it did not correlate in a simple way with target transcription. This is the first report directly linking a discrete genomic function with preferential insertion of a retrotransposon.

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Section: Ty3 Targets Pol Ill-ttanscribed Genesmentioning

confidence: 99%

Ty3 integrates within the region of RNA polymerase III transcription initiation.

Chalker¹,

Sandmeyer²

1992

Genes Dev.

202

187

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“…Numerous studies have indicated that binding of certain proteins to their recognition sequences induces DNA bending and that DNA bending may function in transcriptional activation (16,19,44,47), transcriptional repression (42), DNA replication (1,55,64), and DNA recombination (52,53). We have utilized circular permutation and phasing analysis to test whether binding of ROR␣ isoforms to RORE induces DNA bending.…”

mentioning

confidence: 99%

The Nonconserved Hinge Region and Distinct Amino-Terminal Domains of the RORα Orphan Nuclear Receptor Isoforms are Required for Proper DNA Bending and RORα-DNA Interactions

Mcbroom

Flock

Giguère

1995

Molecular and Cellular Biology

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ROR␣1 and ROR␣2 are two isoforms of a novel member of the steroid-thyroid-retinoid receptor superfamily and are considered orphan receptors since their cognate ligand has yet to be identified. These putative receptors have previously been shown to bind as monomers to a DNA recognition sequence composed of two distinct moieties, a 3 nuclear receptor core half-site AGGTCA preceded by a 5 AT-rich sequence. Recognition of this bipartite hormone response element (RORE) requires both the zinc-binding motifs and a group of amino acid residues located at the carboxy-terminal end of the DNA-binding domain (DBD) which is referred to here as the carboxy-terminal extension. In this report, we show that binding of ROR␣1 and ROR␣2 to the RORE induces a large DNA bend of ϳ130؇ which may be important for receptor function. The overall direction of the DNA bend is towards the major groove at the center of the 3 AGGTCA half-site. The presence of the nonconserved hinge region which is located between the DBD and the putative ligand-binding domain (LBD) or ROR␣ is required for maximal DNA bending. Deletion of a large portion of the amino-terminal domain (NTD) of the ROR␣ protein does not alter the DNA bend angle but shifts the DNA bend center 5 relative to the bend induced by intact ROR␣. Methylation interference studies using the NTD-deleted ROR␣1 mutant indicate that some DNA contacts in the 5 AT-rich half of the RORE are also shifted 5, while those in the 3 AGGTCA half-site are unaffected. These results are consistent with a model in which the ROR␣ NTD and the nonconserved hinge region orient the zinc-binding motifs and the carboxy-terminal extension of the ROR␣ DBD relative to each other to achieve proper interactions with the two halves of its recognition site. Transactivation studies suggest that both protein-induced DNA bending and protein-protein interactions are important for receptor function.The nuclear receptor superfamily encodes a diverse set of transcriptional regulators (7). This superfamily includes receptors for steroids, retinoids, and thyroid hormones as well as a large number of orphan receptors which are structurally and functionally related but whose ligands have not been identified (for references, see reference 28). The domain structures of the receptors are similar in that they each contain four structural regions (11,26). There is an amino-terminal domain (NTD) that is not well conserved among receptors, followed by a highly conserved DNA-binding domain (DBD). The DBD is composed of two class II zinc-binding motifs which fold together to form a single structural unit (52). In some receptors, a group of amino acid residues that extends carboxy terminal to the zinc-binding motifs has also been implicated in DNA binding (31,62,66). These DNA-binding determinants are believed to be involved in both protein-DNA and proteinprotein interactions. The DBD is separated from a moderately conserved ligand-binding domain (LBD) by a hinge region which shows little homology within the nuclear receptor superfamily. The L...

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“…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.…”

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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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FLP Recombinase of the 2 μm circle plasmid of Saccharomyces cerevisiae bends its DNA target

Cited by 50 publications

References 35 publications

Ty3 integrates within the region of RNA polymerase III transcription initiation.

Ty3 integrates within the region of RNA polymerase III transcription initiation.

The Nonconserved Hinge Region and Distinct Amino-Terminal Domains of the RORα Orphan Nuclear Receptor Isoforms are Required for Proper DNA Bending and RORα-DNA Interactions

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

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