Topological selectivity of a hybrid site-specific recombination system with elements from Tn 3 res /resolvase and bacteriophage P1 loxP /Cre a aEdited by M. Yaniv

Kilbride, Elizabeth; Boocock, Martin R.; Stark, W. Marshall

doi:10.1006/jmbi.1999.2864

Cited by 53 publications

(52 citation statements)

References 30 publications

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“…Recombination at psi exclusively yields four-noded catenanes . Intriguingly, the same product topology is obtained when the Xer or res accessory sequences are fused the core recombination site of a nonrelated tyrosine recombinase such as Cre and Flp (Kilbride et al, 1999;Grainge et al, 2000;Gourlay and Col- loms, 2004) This supports the view that both systems use a similar synapse topology to achieve resolution selectivity, and that their accessory elements orient the core recombination sites in a specific configuration. As TnpI uses the same mechanism of strand exchange, the finding that the deletion product between two IRSs is a two-noded catenane suggests that the architecture of the TnpI/IRS complex is different (for further discussion, see Kilbride et al, 2006).…”

Section: A Novel Topological Architecture To Impose Resolution Selectmentioning

confidence: 65%

“…unlinked deletion products or unknotted inversion products; e.g. Kilbride et al, 1999;Grainge et al, 2000;Gourlay and Colloms, 2004), free circles were undetectable in TnpI-catalysed deletion reactions at IR1-IR2 on supercoiled substrates. This suggests that at least one negative supercoil must be trapped or looped out from the substrate to form a productive synapse.…”

Section: Relaxed Recombination and Partial Selectivitymentioning

confidence: 94%

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Self‐control in DNA site‐specific recombination mediated by the tyrosine recombinase TnpI

Vanhooff

Galloy

Agaisse

et al. 2006

Molecular Microbiology

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SummaryTn 4430 is a distinctive transposon of the Tn 3 family that encodes a tyrosine recombinase (TnpI) to resolve replicative transposition intermediates. The internal resolution site of Tn 4430 (IRS, 116 bp) contains two inverted repeats (IR1 and IR2) at the crossover core site, and two additional TnpI binding motifs (DR1 and DR2) adjacent to the core. Deletion analysis demonstrated that DR1 and DR2 are not required for recombination in vivo and in vitro . Their function is to provide resolution selectivity to the reaction by stimulating recombination between directly oriented sites on a same DNA molecule. In the absence of DR1 and/ or DR2, TnpI-mediated recombination of supercoiled DNA substrates gives a mixture of topologically variable products, while deletion between two wild-type IRSs exclusively produces two-noded catenanes. This demonstrates that TnpI binding to the accessory motifs DR1 and DR2 contributes to the formation of a specific synaptic complex in which catalytically inert recombinase subunits act as architectural elements to control recombination sites pairing and strand exchange. A model for the organization of TnpI/IRS recombination complex is presented.

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Section: A Novel Topological Architecture To Impose Resolution Selectmentioning

confidence: 65%

Section: Relaxed Recombination and Partial Selectivitymentioning

confidence: 94%

Self‐control in DNA site‐specific recombination mediated by the tyrosine recombinase TnpI

Vanhooff

Galloy

Agaisse

et al. 2006

Molecular Microbiology

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“…Of particular interest are the entanglement of biopolymers and DNA protein interactions (eg Sumners [47], Bauer et al [5], Colloms et al [13], Grainge et al [24], Kilbride et al [31], Pathania et al [38] and Sumners et al [48]). …”

Section: Biology Background and Experimental Datamentioning

confidence: 99%

“…Mu transposase is involved in transposing the Mu genome within the DNA of the bacterial host. In [38], Pathania et al determined the shape of DNA bound within the Mu transposase protein complex using an experimental technique called difference topology; see Harshey and Jayaram [26; 30], Kilbride et al [31], Grainge et al [24], Pathania et al [38; 39] and Yin et al [59; 60]. Their conclusion was based on the assumption that the DNA is in a branched supercoiled form as described near the end of Section 1 (see Figures 9 and 25).…”

Section: Introductionmentioning

confidence: 99%

Tangle analysis of difference topology experiments: Applications to a Mu protein-DNA complex

Darcy

Luecke

Vázquez

2009

Algebr. Geom. Topol.

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We develop topological methods for analyzing difference topology experiments involving 3-string tangles. Difference topology is a novel technique used to unveil the structure of stable protein-DNA complexes. We analyze such experiments for the Mu protein-DNA complex. We characterize the solutions to the corresponding tangle equations by certain knotted graphs. By investigating planarity conditions on these graphs we show that there is a unique biologically relevant solution. That is, we show there is a unique rational tangle solution, which is also the unique solution with small crossing number. 57M25, 92C40 IntroductionBacteriophage Mu is a virus that infects bacteria. Mu transposase is involved in transposing the Mu genome within the DNA of the bacterial host. In [38], Pathania et al determined the shape of DNA bound within the Mu transposase protein complex using an experimental technique called difference topology; see Harshey and Jayaram [26;30], Kilbride et al [31], Grainge et al [24], Pathania et al [38;39] and Yin et al [59;60]. Their conclusion was based on the assumption that the DNA is in a branched supercoiled form as described near the end of Section 1 (see Figures 9 and 25). We show that this restrictive assumption is not needed, and in doing so, conclude that the only biologically reasonable solution for the shape of DNA bound by Mu transposase is the one found in [38] (shown in Figure 1). We will call this 3-string tangle the PJH solution. The 3-dimensional ball represents the protein complex, and the arcs represent the bound DNA. The Mu-DNA complex modeled by this tangle is called the Mu transpososome.The topological structure of the Mu transpososome will be a key ingredient in determining its more refined molecular structure and in understanding the basic mechanism crystallographic data is combined with the known Tn3 resolvase topological mechanisms of binding and strand-exchange to piece together a more detailed geometrical picture of two resolvase-DNA complexes (Sin and ı ).In Section 1 we provide some biological background and describe eight difference topology experiments from [38]. In Section 2, we translate the biological problem of determining the shape of DNA bound by Mu into a mathematical model. The mathematical model consists of a system of ten 3-string tangle equations ( Figure 11). Using 2-string tangle analysis, we simplify this to a system of four tangle equations ( Figure 24). In Section 3 we characterize solutions to these tangle equations in terms of knotted graphs. This allows us to exhibit infinitely many different 3-string tangle solutions. The existence of solutions different from the PJH solution raises the possibility of alternate acceptable models. In Sections 3-5, we show that all solutions to the mathematical problem other than the PJH solution are too complex to be biologically reasonable, where the complexity is measured either by the rationality or by the minimal crossing number of the 3-string tangle solution.In Section 3, we show that the only rational solution i...

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“…In a Cre recombination complex, the loxP sites align in an antiparallel, essentially planar fashion, and the strand exchange reaction per se does not introduce any DNA crossings (27)(28)(29). Each difference topology assay utilizes a pair of matched substrates that are similar in the arrangement of Mu sites and the location of the loxP sites but differ in the relative loxP orientations; direct in one case and inverted in the other (see the schematics in Fig.…”

Section: R Crosses E-l Four Times Within a Mu Synapse Assembled On Nimentioning

confidence: 99%

The Mu Transposase Interwraps Distant DNA Sites within a Functional Transpososome in the Absence of DNA Supercoiling

Yin

Jayaram

Pathania

et al. 2005

Journal of Biological Chemistry

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A Mu transpososome assembled on negatively supercoiled DNA traps five supercoils by intertwining the left (L) and right (R) ends of Mu with an enhancer element (E). To investigate the contribution of DNA supercoiling to this elaborate synapse in which E and L cross once, E and R twice, and L and R twice, we have analyzed DNA crossings in a transpososome assembled on nicked substrates under conditions that bypass the supercoiling requirement for transposition. We find that the transposase MuA can recreate an essentially similar topology on nicked substrates, interwrapping both E-R and L-R twice but being unable to generate the single E-L crossing. In addition, we deduce that the functional MuA tetramer must contribute to three of the four observed crossings and, thus, to restraining the enhancer within the complex. We discuss the contribution of both MuA and DNA supercoiling to the 5-noded Mu synapse built at the 3-way junction.Many recombinases, including the phage Mu transposase, are strictly dependent on DNA supercoiling for activity. These recombinases and their accessory factors take advantage of both the conformational and thermodynamic properties of supercoiled DNA, which include DNA unwinding, formation of single-stranded regions, extrusion of cruciform structures, sequence-dependent induction of Z-DNA conformation, and DNA bending as well as an increase in the local concentration of DNA sites (1). The present study was undertaken to examine the role of DNA supercoiling in establishing the five-noded topology observed in Mu transposition complexes (2, 3).The Mu transposition pathway is shown in Fig. 1 (4). Two families of DNA sites (high affinity attL and attR sites and lower affinity enhancer (E) 1 sites (Fig. 1A)) are recognized by the transposase MuA through separate DNA binding domains. On the linear phage genome, attL and attR are 37 kilobases apart, whereas the enhancer is 1 kilobase from attL (5). MuA monomers likely first bind the att ends and then interact with the enhancer through bridging contacts. A criss-crossed network of att-enhancer interactions has been deduced (6, 7), an ordered progression of which is thought to result in the series of complexes observed during transposition (Fig. 1B).Mu transposition is strictly dependent on DNA supercoiling of the donor substrate under normal reaction conditions. Several roles for supercoiling have been deciphered. Supercoiling increases the binding affinity of MuA for the L and R ends (8) and of the accessory host factor HU for the L end (9), favors bending at the enhancer (10), facilitates interwrapping of the L, E, and R segments within the Mu synapse (11, 2), and assists "open termini" formation, a rate-limiting step in the assembly of the transpososome (12-16). Supercoiling is not required for the chemical steps of transposition per se (4).During assembly of the five-noded Mu synapse, the enhancer first uniquely interacts with R and interwraps with it twice to form an ER complex (Fig. 1B) (2, 3). In the absence of Escherichia coli HU protein, L i...

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Topological selectivity of a hybrid site-specific recombination system with elements from Tn 3 res /resolvase and bacteriophage P1 loxP /Cre a aEdited by M. Yaniv

Cited by 53 publications

References 30 publications

Self‐control in DNA site‐specific recombination mediated by the tyrosine recombinase TnpI

Self‐control in DNA site‐specific recombination mediated by the tyrosine recombinase TnpI

Tangle analysis of difference topology experiments: Applications to a Mu protein-DNA complex

The Mu Transposase Interwraps Distant DNA Sites within a Functional Transpososome in the Absence of DNA Supercoiling

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