The terminal inverted repeats of IS911: requirements for synaptic complex assembly and activity

Normand, Christophe; Duval‐Valentin, Guy; Haren, Laurence; Chandler, Michaël

doi:10.1006/jmbi.2001.4641

Cited by 31 publications

(76 citation statements)

References 41 publications

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“…Furthermore, protection was comparable to that obtained with individual IRs (indicated schematically in Fig. 2A) (18). Addition of increasing concentrations of unlabeled 150-bp DNA containing either IRR or IRL produced a new DNAprotein complex, b, while complex a disappeared (Fig.…”

Section: Resultssupporting

confidence: 65%

“…2A). Several of these differences are located in the region of the IR recognized by the transposase (18). This raises the possibility that transposase binds differentially to each end.…”

Section: Resultsmentioning

confidence: 99%

“…OrfAB(1-149) was used because, once translated, the full-length OrfAB binds poorly to IS911 ends, presumably as a result of a folding process which masks the DNA binding domain. On the other hand, OrfAB(1-149) assembles specific DNA-protein complexes with IRR and IRL, which we believe reflect bona fide synaptic complexes or transpososomes (11,18). Two radiolabeled fragments containing entire IRL or IRR ends together with some flanking DNA (Materials and Methods) were incubated with increasing concentrations of purified OrfAB(1-149).…”

Section: Resultsmentioning

confidence: 99%

“…It seemed possible that this may reflect a higher affinity of the transposase for IRR than for IRL. Since the full-length transposase, OrfAB, binds poorly to the IRs in vitro (12), we used a truncated derivative, OrfAB(1-149), which exhibits significantly higher binding activity (18). EMSA experiments demonstrated that OrfAB(1-149) assembled an SCB-like complex more efficiently when the target DNA carried IRR rather than IRL.…”

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confidence: 99%

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Bias between the Left and Right Inverted Repeats during IS911Targeted Insertion

et al. 2008

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IS911 is a bacterial insertion sequence composed of two consecutive overlapping open reading frames (ORFs [orfA and orfB]) encoding the transposase (OrfAB) as well as a regulatory protein (OrfA). These ORFs are bordered by terminal left and right inverted repeats (IRL and IRR, respectively) with several differences in nucleotide sequence. IS911 transposition is asymmetric: each end is cleaved on one strand to generate a free 3-OH, which is then used as the nucleophile in attacking the opposite insertion sequence (IS) end to generate a free IS circle. This will be inserted into a new target site. We show here that the ends exhibit functional differences which, in vivo, may favor the use of one compared to the other during transposition. Electromobility shift assays showed that a truncated form of the transposase [OrfAB(1-149)] exhibits higher affinity for IRR than for IRL. While there was no detectable difference in IR activities during the early steps of transposition, IRR was more efficient during the final insertion steps. We show here that the differential activities between the two IRs correlate with the different affinities of OrfAB(1-149) for the IRs during assembly of the nucleoprotein complexes leading to transposition. We conclude that the two inverted repeats are not equivalent during IS911 transposition and that this asymmetry may intervene to determine the ordered assembly of the different protein-DNA complexes involved in the reaction.

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Section: Resultssupporting

confidence: 65%

“…2A). Several of these differences are located in the region of the IR recognized by the transposase (18). This raises the possibility that transposase binds differentially to each end.…”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Bias between the Left and Right Inverted Repeats during IS911Targeted Insertion

et al. 2008

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“…In this case, the molecular assembly is dimeric: each doublestranded DNA molecule is bound to both Tnp subunits (8). In other cases, where no crystallographic data are available, the exact stoichiometry of the complex has yet to be defined, as explained for IS911 (21). No complete synaptic complex has so far been crystallized for the eukaryotic transposons, but the structure of Tnp/ITR complexes has been analyzed by a biochemical approach, as in the case of Sleeping Beauty, a Tc1-like element (6, 13).…”

mentioning

confidence: 99%

Assembly of the mariner Mos1 Synaptic Complex

Augé-Gouillou

Brillet

Hamelin

et al. 2005

Molecular and Cellular Biology

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The mobility of transposable elements via a cut-and-paste mechanism depends on the elaboration of a nucleoprotein complex known as the synaptic complex. We show here that the Mos1 synaptic complex consists of the two inverted terminal repeats of the element brought together by a transposase tetramer and is designated paired-end complex 2 (PEC2). The assembly of PEC2 requires the formation of a simpler complex, containing one terminal repeat and two transposase molecules and designated single-end complex 2 (SEC2). In light of the formation of SEC2 and PEC2, we demonstrate the presence of two binding sites for the transposase within a single terminal repeat. We have found that the sequence of the Mos1 inverted terminal repeats contains overlapping palindromic and mirror motifs, which could account for the binding of two transposase molecules "side by side" on the same inverted terminal repeat. We provide data indicating that the Mos1 transposase dimer is formed within a single terminal repeat through a cooperative pathway. Finally, the concept of a tetrameric synaptic complex may simply account for the inability of a single mariner transposase molecule to interact at the same time with two kinds of DNA: the inverted repeat and the target DNA.Mos1 is an autonomous mariner transposable element first isolated from Drosophila mauritiana. It is 1,286 bp long, with 28-bp imperfect inverted terminal repeats (ITR), and contains a single open reading frame encoding a 345-amino-acid transposase (Tnp). On the basis of the sequence of the Tnp and the organization of the element, mariner has been grouped together with the Tc1 and pogo transposable elements to form the Tc1/mariner superfamily (27). One of the main properties of the Tc1/mariner elements is that they do not require host factors for their mobility, because the Tnp is sufficient to promote all the transposition steps. This property accounts for the wide distribution of the Tc1/mariner superfamily among eukaryotic organisms and makes it possible to develop DNA transfer vectors based on Tc1/mariner elements (22).mariner transposes by a cut-and-paste mechanism, similar to that described for related bacterial insertion sequences (4). Briefly, the two ends of the elements are brought together by transposase oligomerization to form a synaptic complex that triggers cleavages at the transposon ends. This complex is usually designated a paired-end complex (PEC). The Tnp then promotes the integration of the excised transposon at a new target site. Assembling the highly organized synaptic complex is the key of transposition, in which the cleavages progress step by step. The structure of this synaptic complex has been elucidated for several prokaryotic transposons, such as Tn5. In this case, the molecular assembly is dimeric: each doublestranded DNA molecule is bound to both Tnp subunits (8). In other cases, where no crystallographic data are available, the exact stoichiometry of the complex has yet to be defined, as explained for IS911 (21). No complete synaptic complex has s...

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Assembly of the Tc1 and mariner transposition initiation complexes depends on the origins of their transposase DNA binding domains

2006

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In this review, we focus on the assembly of DNA/protein complexes that trigger transposition in eukaryotic members of the IS630-Tc1-mariner (ITm) super-family, the Tc1- and mariner-like elements (TLEs and MLEs). Elements belonging to this super-family encode transposases with DNA binding domains of different origins, and recent data indicate that the chimerization of functional domains has been an important evolutionary aspect in the generation of new transposons within the ITm super-family. These data also reveal that the inverted terminal repeats (ITRs) at the ends of transposons contain three kinds of motif within their sequences. The first two are well known and correspond to the cleavage site on the outer ITR extremities, and the transposase DNA binding site. The organization of ITRs and of the transposase DNA binding domains implies that differing pathways are used by MLEs and TLEs to regulate transposition initiation. These differences imply that the ways ITRs are recognized also differ leading to the formation of differently organized synaptic complexes. The third kind of motif is the transposition enhancers, which have been found in almost all the functional MLEs and TLEs analyzed to date. Finally, in vitro and in vivo assays of various elements all suggest that the transposition initiation complex is not formed randomly, but involves a mechanism of oriented transposon scanning.

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The terminal inverted repeats of IS911: requirements for synaptic complex assembly and activity

Cited by 31 publications

References 41 publications

Bias between the Left and Right Inverted Repeats during IS911Targeted Insertion

Bias between the Left and Right Inverted Repeats during IS911Targeted Insertion

Assembly of the mariner Mos1 Synaptic Complex

Assembly of the Tc1 and mariner transposition initiation complexes depends on the origins of their transposase DNA binding domains

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