Site-specific recombination by Tn3 resolvase: Topological changes in the forward and reverse reactions

Stark, W. Marshall; Sherratt, David J.; Boocock, Martin R.

doi:10.1016/0092-8674(89)90111-6

Cited by 182 publications

(168 citation statements)

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“…Recombination leads to loss of stored negative superhelicity, the energy of which is assumed to drive the strand exchange. Determination of the resultant ΔLk, by sharply limiting the acceptable mechanisms of strand exchange, facilitated the identification of the structure of synaptic complexes (Stark et al 1989;Kanaar et al 1989;Crisona et al 1999;Canosa et al 2003). In that sense, analyses of topological constraints imposed on intramolecular reactions catalyzed by the invertase/resolvase family of recombinases turned out to be most informative.…”

Section: H-ns Nucleoprotein Complexesmentioning

confidence: 99%

The regulatory role of DNA supercoiling in nucleoprotein complex assembly and genetic activity

Muskhelishvili

Travers

2016

Biophys Rev

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We argue that dynamic changes in DNA supercoiling in vivo determine both how DNA is packaged and how it is accessed for transcription and for other manipulations such as recombination. In both bacteria and eukaryotes, the principal generators of DNA superhelicity are DNA translocases, supplemented in bacteria by DNA gyrase. By generating gradients of superhelicity upstream and downstream of their site of activity, translocases enable the differential binding of proteins which preferentially interact with respectively more untwisted or more writhed DNA. Such preferences enable, in principle, the sequential binding of different classes of protein and so constitute an essential driver of chromatin organization.Keywords DNA supercoiling . DNA translocases . Chromatin organization . Superhelicity gradients . DNA untwisting . DNAwrithing Dynamic changes of DNA supercoiling act as a driving force behind the alterations of genetic activity and DNA compaction both in eukaryotes and prokaryotes. Both these processes involve formation of spatially organized nucleoprotein structures by DNA architectural proteins. Whereas in eukaryotes the paradigmal example is the histone octamer, in prokaryotes a similar function is attributed to a small class of highly abundant nucleoid-associated proteins. We argue that, depending on the primary sequence organization, the changes of superhelicity elicit various alterations of local DNA geometry, while these, in turn, facilitate the assembly of distinct nucleoprotein structures pertinent to genetic function. Genome-wide conversion of the superhelical energy into various three-dimensional DNA structures thus acts as an analogue code coordinating the energy supply with the genetic expression of the chromosome.Recent studies make it increasingly clear that the DNA double helix carries at least two types of encoded information and that these include the well-known genetic code and the structural information determining the form and physical properties of the double helix itself. Since both these information types are inscribed in the same DNA sequence, and yet one is discrete whereas the other is continuous, they have been respectively dubbed the digital and analog DNA codes (Marr et al. 2008;Travers et al. 2012;Muskhelishvili and Travers 2013). The potential of the DNA to adopt distinct configurations is strongly modulated by DNA supercoiling, which is increasingly recognized as one of the major forces coordinating the cellular DNA transactions in both prokaryotes (including Archaea) and eukaryotes.DNA supercoiling can be generated by the simple application of torque to the double helix (Strick et al. 1998) but, importantly, because the DNA molecule itself possesses chirality-it is, after all, a right-handed double helix-the local structures stabilized by changes in superhelical density depend on both the sign and strength of the applied torque. For example, both positive and negative torque (respectively overand underwinding of the double helix) can change the intrinsic coiling of ...

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Section: H-ns Nucleoprotein Complexesmentioning

confidence: 99%

The regulatory role of DNA supercoiling in nucleoprotein complex assembly and genetic activity

Muskhelishvili

Travers

2016

Biophys Rev

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“…The crystal structure of ␥␦ resolvase bound to a cleavage site reveals a unique arrangement of the catalytic and DNA-binding domains in that they bind to different faces of the helix (8). Although two models have been proposed (9)(10)(11), the structure of the synapse and the changes in the conformation of resolvase that bring about strand exchange are still a mystery (12).…”

mentioning

confidence: 99%

“…The sequences are not symmetrical and therefore sites can be in direct or inverse orientation. The reactions catalyzed by Tn3 resolvase and Gin, however, are topologically defined, with precise arrangements of the components in the synaptic complex and in the strand-exchange reaction itself (2,9,10,22,23). Thus, in the resolvase system only res sites that are in direct repeat recombine, and in the Gin system only inverted sites recombine; recombination is totally blocked if a synapse with the ''wrong topology'' is formed because of sites being in the incorrect orientation.…”

mentioning

confidence: 99%

In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family

Thorpe

Smith

1998

Proc. Natl. Acad. Sci. U.S.A.

411

353

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The genome of the broad host range Streptomyces temperate phage, C31, is known to integrate into the host chromosome via an enzyme that is a member of the resolvase͞invertase family of site-specific recombinases. The recombination properties of this novel integrase on the phage and Streptomyces ambofaciens attachment sites, attP and attB, respectively, were investigated in the heterologous host, Escherichia coli, and in an in vitro assay by using purified integrase. The products of attP͞B recombination, i.e., attL and attR, were identical to those obtained after integration of the prophage in S. ambofaciens. In the in vitro assay only buffer, purified integrase, and DNAs encoding attP and attB were required. Recombination occurred irrespective of whether the substrates were supercoiled or linear. A mutant integrase containing an S12F mutation was completely defective in recombination both in E. coli and in vitro. No recombination was observed between attB͞attB, attP͞attP, attL͞R, or any combination of attB or attP with attL or attR, suggesting that excision of the prophage (attL͞R recombination) requires an additional phage-or Streptomyces-encoded factor. Recombination could occur intramolecularly to cause deletion between appropriately orientated attP and attB sites. The results show that directionality in C31 integrase is strictly controlled by nonidentical recombination sites with no requirement to form the topologically defined structures that are more typical of the resolvases͞invertases.In site-specific recombination a recombinase interacts with a specific site in the DNA, brings the sites together in a synapse, and then catalyzes strand exchange so that the DNA is cleaved and religated to opposite partners (1, 2). The reaction can result in integration, inversion, or resolution͞excision depending on the position and orientation of the recombination sites, their interactions with recombinase, and the presence or absence of accessory factors or sites. Site-specific recombinases in bacteria fall into one of two very distinct families, the integrase-like enzymes and the resolvase͞invertases, on the basis of amino acid sequence similarities and their different mechanisms of catalysis (1-3). Recombination by members of the integrase family (e.g., integrase, P1 Cre-loxP) is well understood and involves the formation and resolution of a Holliday junction intermediate during which the DNA is transiently attached to the enzyme through a phosphotyrosine linkage (4-6). The resolvase͞invertase family of enzymes (e.g., Tn3 or ␥␦ resolvases, Mu Gin invertase) act via a concerted, four-strand staggered break and rejoining mechanism during which a phosphoserine linkage is formed between the enzyme and the DNA (2, 7). The crystal structure of ␥␦ resolvase bound to a cleavage site reveals a unique arrangement of the catalytic and DNA-binding domains in that they bind to different faces of the helix (8). Although two models have been proposed (9-11), the structure of the synapse and the changes in the conformation of...

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“…The IllOR and V114R mutants were constructed to disrupt helix dimerization of the catalytic domain and, as expected, were found to behave as monomers at the concentrations used in gel mobility shift assays and gel filtration analysis (Hughes et al, 1993). The monomeric form of resolvase is likely to represent a functional intermediate in the strand exchange process according to the subunit rotation model (Stark et al, 1989(Stark et al, , 1991. NMR analysis was performed for the domains in the I1 10R resolvase and a comparative structural characterization of the monomeric protein is presented.…”

mentioning

confidence: 63%

“…The subunit rotation model for strand exchange (Stark et al, 1989) is the most direct interpretation accounting for the topological changes occumng during recombination. In this model, a pair of subunits on the left side of a covalently linked tetramer makes a 180" right-handed rotation relative to the subunits on the right side.…”

Section: Importance Of Flexibility In the Catalytic Domainmentioning

confidence: 99%

Secondary and tertiary structural changes in γδ resolvase: Comparison of the wild‐type enzyme, the I11OR mutant, and the C‐terminal DNA binding domain in solution

et al. 1997

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y6Resolvase is a site-specific DNA recombinase (MI 20.5 kDa) in Escherichia coli that shares homology with a family of bacterial resolvases and invertases. We have characterized the secondary and tertiary structural behavior of the cloned DNA binding domain (DBD) and a dimerization defective mutant in solution. Low-salt conditions were found to destabilize the tertiary structure of the DBD dramatically, with concomitant changes in the secondary structure that were localized near the hinge regions between the helices. The molten tertiary fold appears to contribute significantly to productive DNA interactions and supports a mechanism of DNA-induced folding of the tertiary structure, a process that enables the DBD to adapt in conformation for each of the three imperfect palindromic sites. At high salt concentrations, the monomeric I1 1OR resolvase shows a minimal perturbation to the three helices of the DBD structure and changes in the linker segment in comparison to the cloned DBD containing the linker. Comparative analysis of the NMR spectra suggest that the I1 1OR mutant contains a folded catalytic core of -60 residues and that the segment from residues 100 to 149 are devoid of regular structure in the IllOR resolvase. No increase in the helicity of the linker region of IllOR resolvase occurs on binding DNA. These results support a subunit rotation model of strand exchange that involves the partial unfolding of the catalytic domains.Keywords: DNA; folding; interaction; NMR; protein; recombination; resolvase; structure y6 Resolvase (MI 20.5 kDa) is a site-specific DNA recombinase in Escherichia coli that shares homology with a family of resolvases and invertases in a variety of bacteria (Grindley & Reed, 1985). The structure of resolvase can be divided into three distinct regions, consisting of a catalytic domain (residues 1-120), a DNA binding domain (residues 149-183), and a linker region between the two globular domains. Limited proteolysis of 715 resolvase yields a C-terminal 43-residue fragment (5 kDa) with DNA binding activity and an N-terminal 140-residue fragment (15.5 kDa) with oligomerization and catalytic functions. The structure of the catalytic domain has been determined by X-ray crystallography

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Site-specific recombination by Tn3 resolvase: Topological changes in the forward and reverse reactions

Cited by 182 publications

References 34 publications

The regulatory role of DNA supercoiling in nucleoprotein complex assembly and genetic activity

The regulatory role of DNA supercoiling in nucleoprotein complex assembly and genetic activity

In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family

Secondary and tertiary structural changes in γδ resolvase: Comparison of the wild‐type enzyme, the I11OR mutant, and the C‐terminal DNA binding domain in solution

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