Half-att site substrates reveal the homology independence and minimal protein requirements for productive synapsis in λ excisive recombination

Nunes-Düby, Simone E.; Matsumoto, Lloyd; Landy, Arthur

doi:10.1016/0092-8674(89)90881-7

Cited by 65 publications

(56 citation statements)

References 34 publications

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“…5B), similar to the results above when an attR containing a biotin substitution in P2 is recombined with an attL containing a biotin insertion in PЈ3. 3 These results demonstrate that the physical DNA requirements for excisive recombination extend no further than the P2 site in attR and the PЈ2 site in attL, consistent with earlier mutational analyses (15,46,47,54).…”

Section: Dna In This Region (Nc Resectsupporting

confidence: 74%

A Biotin Interference Assay Highlights Two Different Asymmetric Interaction Profiles for λ Integrase Arm-type Binding Sites in Integrative Versus Excisive Recombination

Hazelbaker

Azaro

Landy

2008

Journal of Biological Chemistry

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The site-specific recombinase integrase encoded by bacteriophage promotes integration and excision of the viral chromosome into and out of its Escherichia coli host chromosome through a Holliday junction recombination intermediate. This intermediate contains an integrase tetramer bound via its catalytic carboxyl-terminal domains to the four "core-type" sites of the Holliday junction DNA and via its amino-terminal domains to distal "arm-type" sites. The two classes of integrase binding sites are brought into close proximity by an ensemble of accessory proteins that bind and bend the intervening DNA. We have used a biotin interference assay that probes the requirement for major groove protein binding at specified DNA loci in conjunction with DNA protection, gel mobility shift, and genetic experiments to test several predictions of the models derived from the x-ray crystal structures of minimized and symmetrized surrogates of recombination intermediates lacking the accessory proteins and their cognate DNA targets. Our data do not support the predictions of "non-canonical" DNA targets for the N-domain of integrase, and they indicate that the complexes used for x-ray crystallography are more appropriate for modeling excisive rather than integrative recombination intermediates. We suggest that the difference in the asymmetric interaction profiles of the N-domains and arm-type sites in integrative versus excisive recombinogenic complexes reflects the regulation of recombination, whereas the asymmetry of these patterns within each reaction contributes to directionality. Integrase (Int)2 is a site-specific recombinase encoded by bacteriophage that promotes integration and excision of the viral chromosome into and out of the chromosome of the Escherichia coli host (1, 2). Int-mediated recombination between attP and attB on the and bacterial chromosomes, respectively, generates an integrated prophage bounded by attL and attR, which are also partners for the excisive recombination that reforms attP and attB (1, 3). Int is a member of a large family of tyrosine recombinases that execute site-specific recombination in the absence of high energy cofactors via a sequential pair of staggered (by seven base pairs, in the case of Int) single-strand exchanges that first generate and then resolve a four-way DNA junction known as a Holliday junction (HJ, see Fig. 1A) (4 -7). Each DNA strand cleavage involves nucleophilic attack by an active site tyrosine (Tyr-342) to form a high energy covalent 3Ј-phosphotyrosine bond. Attack of this intermediate by the 5Ј-hydroxyl of the swapped DNA strand forms one of the four ligated novel joints (8).Int is a well studied member of the subgroup of heterobivalent recombinases that bind and bridge two different families of DNA sequences (9). The amino-terminal domain of Int (N-domain, residues 1-63) binds with high affinity to arm-type Int binding sites (P1, P2, PЈ1, PЈ2, and PЈ3) that are distant from the core region where DNA strand exchange occurs (10) (Fig. 1A). Each of the core-type Int binding...

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Section: Dna In This Region (Nc Resectsupporting

confidence: 74%

A Biotin Interference Assay Highlights Two Different Asymmetric Interaction Profiles for λ Integrase Arm-type Binding Sites in Integrative Versus Excisive Recombination

Hazelbaker

Azaro

Landy

2008

Journal of Biological Chemistry

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“…(24). We therefore adapted the half-site assay described by Landy and coworkers for the lambda Int recombination system (22,23). The basis of this assay is that if FLP catalyzes strand scission of a half-FRT site, the oligonucleotide that is generated is lost by diffusion, leaving the FLP protein covalently bound to the half-site via a phosphotyrosine bond (12) (Fig.…”

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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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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“…To do this, they rely on an asymmetric 6-to 8-bp "overlap" sequence at the center of the crossover site . The alignment is not tested during synapsis of the sites but during the strand exchange reaction, in which a Holliday junction migrates through the asymmetric core sequence (de Massy et al 1989;Nash and Robertson 1989;Nunes-Duby et al 1989). The Tn3 res crossover site, however, is cut with only a 2-bp stagger at the center of a perfectly symmetrical hexanucleotide, TTATAA (Reed and Grindley 1981).…”

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confidence: 99%

Determinants of correct res site alignment in site-specific recombination by Tn3 resolvase.

Bednarz¹,

Boocock²,

Sherratt³

1990

Genes & Development

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In site-specific recombination reactions catalyzed by Tn3 resolvase, the right and left arms of the res site are always religated to the correct partner. This poses the problem of how resolvase aligns the two sites correctly for the cleavage/religation reaction. We show that the "accessory" binding subsites II and III of res are important for correct alignment of the adjoining crossover subsite (subsite I). Deletion of subsites II and III from one of the two res sites removes a barrier to recombination between incorrectly aligned crossover subsites. Correct alignment does not require any DNA sequence asymmetry in the crossover subsite, DNA supercoiling, or covalent linkage of the two res sites. Our results suggest that correct subsite I alignment is determined by local, resolvase-mediated interactions of subsites II and III of both partners, consistent with a current model of the synapse. Surprisingly, the topological selectivity for intramolecular resolution in a supercoiled substrate does not require subsites II and III in both recombination partners.

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Half-att site substrates reveal the homology independence and minimal protein requirements for productive synapsis in λ excisive recombination

Cited by 65 publications

References 34 publications

A Biotin Interference Assay Highlights Two Different Asymmetric Interaction Profiles for λ Integrase Arm-type Binding Sites in Integrative Versus Excisive Recombination

A Biotin Interference Assay Highlights Two Different Asymmetric Interaction Profiles for λ Integrase Arm-type Binding Sites in Integrative Versus Excisive Recombination

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

Determinants of correct res site alignment in site-specific recombination by Tn3 resolvase.

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