Enhancer-independent mutants of the Cin recombinase have a relaxed topological specificity.

Haffter, P.; Bickle, Thomas A.

doi:10.1002/j.1460-2075.1988.tb03287.x

Cited by 64 publications

(53 citation statements)

References 30 publications

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“…Recent experiments with Tn3 resolvase have implicated interwrapping at the enhancer in determining the topological selectivity of the reaction (4); enhancer-independent resolvase mutants simultaneously lost their normal specificity for directly oriented sites and for supercoiled substrates. Similar results have been reported with the Gin invertase family (5,6). Since the Mu experiments implicating DNA topology in determining the orientation specificity of the ends (2) were done before the discovery of the enhancer, one objective of the present study was to dissect the contribution of the enhancer, if any, to the topological filter.…”

supporting

confidence: 73%

The Mu Enhancer Is Functionally Asymmetric Both incis and in trans

Jiang

Harshey

2001

Journal of Biological Chemistry

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Mu DNA transposition from a negatively supercoiled DNA substrate requires interaction of an enhancer element with the left (attL) and right (attR) ends of Mu. The orientation of the L and R ends with respect to each other (inverted) and with respect to the enhancer is normally inviolate. We show that when the enhancer is provided in trans as a linear fragment, the head to head orientation of the L/R ends is still required. Each functional half of the linear enhancer maintains the same "cross-wise" interaction with the subsites L1 and R1, when present in cis or in trans. In reactions catalyzed by an enhancer-independent variant of the Mu transposase, the need for negative supercoiling of the substrate and the inverted orientation of L and R ends is not relaxed. These results show that the orientation specificity of the enhancer is not determined by its topological linkage to the Mu ends. There is a functional asymmetry inherent to the enhancer. Furthermore, the enhancer does not directly impose topological constraints on the transposition reaction or specify the reactive orientation of the Mu ends.Site-specific recombination systems in both prokaryotes and eukaryotes require interaction of specific DNA sequences. Although some systems can carry out recombination with the interacting recombination sites in any orientation (e.g. phage integration/excision, phage P1 Cre/lox recombination, yeast 2-m plasmid Flp/FRT recombination), other systems have a strict requirement for just one orientation (e.g. Mu transposition, Tn3/␥␦ resolvase reactions, Hin/Gin inversion reactions) (see Ref. 1). A hallmark of the latter systems is the employment of enhancers (or accessory DNA sites) and the requirement for negative DNA supercoiling. Either one or both of these elements (enhancers and DNA supercoiling) could contribute to the orientation specificity of the recombination sites in these systems. For the Mu transposition system, the role of supercoiling was explored by Craigie and Mizuuchi (2) prior to the discovery of the enhancer. Transposition of the L and R ends of Mu present in two different orientations on catenated or knotted supercoiled DNA substrates was monitored. The pattern of reactivity of Mu ends on these novel substrates led the authors to suggest that the normally inverted L and R ends might juxtapose most easily in an "intertwined" parallel configuration on negatively supercoiled DNA; for directly oriented sites an energy barrier must be overcome to bring them together in this parallel configuration. Thus, DNA topology was responsible for sensing the relative orientation of the Mu ends. Experiments with Tn3 resolvase were consistent with the Mu results in suggesting the existence of a "topological filter" (defined as a combined effect of constraints derived from both the circularity of the DNA substrate and the requirement for a highly specific interwrapping of the recombining sites; see Ref. 1) that limits productive interaction to a particular orientation of sites (3). Recent experiments with Tn3 resolvase hav...

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supporting

confidence: 73%

The Mu Enhancer Is Functionally Asymmetric Both incis and in trans

Jiang

Harshey

2001

Journal of Biological Chemistry

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“…This region is also responsible for interactions with the minor groove at the DNA cognate site (Hughes et al 1990;Yang & Steitz 1995;reviewed in Grindley 1994). Interestingly, however, the same region in invertases, in addition seems to be either directly or indirectly involved in mediating protein-protein interactions with their host factor, Fis (Klippel et al 1988;Haffter & Bickle 1988;Deufel et al 1997).…”

Section: Resultsmentioning

confidence: 99%

“…N106 and F114, differ from those found in the wild-type and mutant Gin. Interestingly, a change of histidine to asparagine at position 106 inactivates Cin invertase (Haffter & Bickle 1988). The latter residue is found at the corresponding position in ISXc5 resolvase.…”

Section: Discussionmentioning

confidence: 98%

The resolvase encoded by Xanthomonas campestris transposable element ISXc5 constitutes a new subfamily closely related to DNA invertases

Liu

Hühne

et al. 1998

Genes to Cells

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Background: Conservative site-specific recombination is responsible for the resolution of cointegrates which result during the transposition of class II transposable elements. Resolution is catalysed by a transposonencoded recombinase, resolvase, that belongs to a large family of recombinases, including DNA invertases. Resolvases and the related invertases are likely to employ similar reaction mechanisms during recombination. There are important differences, however. Resolvases require two accessory DNA binding sites within each of the two directly repeated recombination sites. Invertases instead need a host factor, Fis, and an enhancer type DNA sequence, in addition to two inversely orientated recombination sites.

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“…1). In the natural systems, the orientation of recombination sites has similar determinative function (29)(30)(31), although accessory functions have evolved in many cases to restrict the secondary activities [e.g., FIS enhances inversion by Gin-invertase (32)]. …”

Section: Discussionmentioning

confidence: 99%

“…4B). Truncated IS30 Tpase carries out secondary reactions similarly to natural systems: Serinvertases cause deletion or cointegration (30,31,34) and can integrate into secondary sites (35). More accurate functioning can be achieved by natural selection, as shown in the presented model (Fig.…”

Section: Discussionmentioning

confidence: 99%

Site-specific recombination by the DDE family member mobile element IS30transposase

Kiss

Szabó

Olasz

2003

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

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DNA rearrangements carried out by site-specific recombinases and transposases (Tpases) show striking similarities despite the wide spectrum of the catalytic mechanisms involved in the reactions. Here, we show that the bacterial insertion sequence (IS)30 element can act similarly to site-specific systems. We have developed an inversion system using IS30 Tpase and a viable phage, where the integration͞excision system is replaced with IS30. Both models have been proved to operate analogously to their natural counterpart, confirming that a DDE family Tpase is able to fulfill the functions of site-specific recombinases. This work demonstrates that distinction between transposition and site-specific recombination becomes blurred, because both functions can be fulfilled by the same enzyme, and both types of rearrangements can be achieved by the same catalytic mechanisms.I t is now well documented that genetic information can be reshuffled by mobile elements that are usually distinguished as site-specific systems and transposons. Even though the output of their activity can be simplified to insertion, deletion, or inversion of DNA segments, transposition and site-specific recombination are generally regarded as distinct phenomena (1). Classification according to the protein-sequence motifs revealed that some transposases (Tpases) and site-specific recombinases (Recases) with entirely different functions fall within the same family (2-6). This finding supports that mechanistically dissimilar enzymes can perform similar biological functions and suggests that transposition and site-specific recombination may be closer to each other in some respect than was supposed earlier. Further support for this idea may emerge from insertion sequence (IS) elements that form an active junction composed of the inverted repeats (IRs) (7-10). This IR-IR junction shows similarities in its structure and function to the recombination sites of sitespecific systems.The Escherichia coli element, IS30, encoding a Tpase with the well conserved DDE signature (11), belongs to the increasing class of elements that transpose through an intermediate formed by abutted IRs (8). The IR-IR joint bracketing a 2-bp spacer is composed of the 26-bp left and right IR ends (IRL and IRR, respectively) that differ at three positions (12). IR-IR joints occur in both IS dimers and minicircles that can integrate into hot spot sequences or next to other IS30 ends (13-15). Whereas transposition into a hot spot is a conventional way of translocation, which accompanies the resolution of IR-IR junction, targeting an IR end seems more specific for IS30 and generates a new IR-IR joint. The IRs, together with the adjacent IS sequences, are important for the directionality of recombination, because, as always, IRL-IRR junction arises through dimerization or targeting an IR (8, 13, 16). The most frequent reaction has been observed when both donor and target DNA contain an IR-IR junction (13). In this case, the rearrangement is remarkably reversible and resembles site-specific ...

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Enhancer-independent mutants of the Cin recombinase have a relaxed topological specificity.

Cited by 64 publications

References 30 publications

The Mu Enhancer Is Functionally Asymmetric Both incis and in trans

The Mu Enhancer Is Functionally Asymmetric Both incis and in trans

The resolvase encoded by Xanthomonas campestris transposable element ISXc5 constitutes a new subfamily closely related to DNA invertases

Site-specific recombination by the DDE family member mobile element IS30transposase

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