Sub-terminal Sequences Modulating IS30 Transposition in Vivo and in Vitro

Szabó, Márton; Kiss, János; Nagy, Zita; Chandler, Michaël; Olasz, Ferenc

doi:10.1016/j.jmb.2007.10.043

Cited by 25 publications

(54 citation statements)

References 32 publications

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“…(i) Transposase producer and target plasmids. The transposase producer was p15A-based Km r plasmid pJKI324 carrying IS30 Orf-A under the control of the tac promoter (33). For the in vitro assays, the full-length Tpase was purified from strain ER2566 (New England Biolabs) harboring pJKI380 (33).…”

Section: Methodsmentioning

confidence: 99%

“…This process involves the Tpasecatalyzed cleavage of one strand at the 3Ј IS end, which then attacks the same strand 2 bp outside the other IR. This strand transfer generates a single-strand bridge between the ends and leads to a figure-eight structure (33). This active transposition intermediate carrying the joined IRs probably proceeds via replicative resolution, as described for IS911 (11,25) and IS2 (16).…”

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confidence: 94%

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Functional Organization of the Inverted Repeats of IS30

2010

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The mobile element IS30 has 26-bp imperfect terminal inverted repeats (IRs) that are indispensable for transposition. We have analyzed the effects of IR mutations on both major transposition steps, the circle formation and integration of the abutted ends, characteristic for IS30. Several mutants show strikingly different phenotypes if the mutations are present at one or both ends and differentially influence the transposition steps. The two IRs are equivalent in the recombination reactions and contain several functional regions. We have determined that positions 20 to 26 are responsible for binding of the N-terminal domain of the transposase and the formation of a correct 2-bp spacer between the abutted ends. However, integration is efficient without this region, suggesting that a second binding site for the transposase may exist, possibly within the region from 4 to 11 bp. Several mutations at this part of the IRs, which are highly conserved in the IS30 family, considerably affected both major transposition steps. In addition, positions 16 and 17 seem to be responsible for distinguishing the IRs of related insertion sequences by providing specificity for the transposase to recognize its cognate ends. Finally, we show both in vivo and in vitro that position 3 has a determining role in the donor function of the ends, especially in DNA cleavage adjacent to the IRs. Taken together, the present work provides evidence for a more complex organization of the IS30 IRs than was previously suggested.

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Section: Methodsmentioning

confidence: 99%

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confidence: 94%

Functional Organization of the Inverted Repeats of IS30

2010

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“…The founding member of the family, IS30, is the best characterized at the mechanistic level (117,(182)(183)(184)(185)(186)(187)(188)(189) and an in vitro transposition system has been developed (190). This 1,221 bp long Escherichia coli element belongs to a growing class of IS known to transpose through an intermediate formed by abutting the IR, donor primed transposon replication.…”

Section: (Iii) Mechanism and Insertion Specificitymentioning

confidence: 99%

“…IR-IR junctions have also been detected in some other IS30 family members such as IS18 (181), IS4351 (191), and IS1470 (193). A structure in which two IS30 ends are linked by a single-strand bridge (forming a figure of eight structure on a circular plasmid, has been identified (190).…”

Section: (Iii) Mechanism and Insertion Specificitymentioning

confidence: 99%

Everyman's Guide to Bacterial Insertion Sequences

et al. 2015

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The number and diversity of known prokaryotic insertion sequences (IS) have increased enormously since their discovery in the late 1960s. At present the sequences of more than 4000 different IS have been deposited in the specialized ISfinder database. Over time it has become increasingly apparent that they are important actors in the evolution of their host genomes and are involved in sequestering, transmitting, mutating and activating genes, and in the rearrangement of both plasmids and chromosomes. This review presents an overview of our current understanding of these transposable elements (TE), their organization and their transposition mechanism as well as their distribution and genomic impact. In spite of their diversity, they share only a very limited number of transposition mechanisms which we outline here. Prokaryotic IS are but one example of a variety of diverse TE which are being revealed due to the advent of extensive genome sequencing projects. A major conclusion from sequence comparisons of various TE is that frontiers between the different types are becoming less clear. We detail these receding frontiers between different IS-related TE. Several, more specialized chapters in this volume include additional detailed information concerning a number of these.In a second section of the review, we provide a detailed description of the expanding variety of IS, which we have divided into families for convenience. Our perception of these families continues to evolve and families emerge regularly as more IS are identified. This section is designed as an aid and a source of information for consultation by interested specialist readers.

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“…However, some transposons carry arrays of sequence elements which differ at each end, and these differences result in a functional asymmetry between the extremities (5). Different activities between the left and right IRs (IRL and IRR, respectively) have also been observed in several of the simplest ISs: for IS10 (Tn10), binding of the integration host factor and H-NS host proteins to subterminal sites close to the outside end plays an important role in regulating transposition (3,4,27,35); the two ends of IS50 also differ in sequence and in activity during Tn5 transposition (7,29), as do those of IS30 (28). In the case of Mos1, the transposase binds preferentially to the right end, which differs in four positions from the left (1,2,36).…”

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confidence: 99%

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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Sub-terminal Sequences Modulating IS30 Transposition in Vivo and in Vitro

Cited by 25 publications

References 32 publications

Functional Organization of the Inverted Repeats of IS30

Functional Organization of the Inverted Repeats of IS30

Everyman's Guide to Bacterial Insertion Sequences

Bias between the Left and Right Inverted Repeats during IS911Targeted Insertion

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