Identification and Characterization of the Linear IS3 Molecules Generated by Staggered Breaks

IS150, a member of the widespread IS3 family, contains two consecutive out-of-phase open reading frames, orfA and orfB, that partially overlap. These open reading frames encode three proteins, InsA, InsB, and the InsAB protein, which is jointly encoded by both open reading frames by means of programmed translational frameshifting. We demonstrate that the InsAB protein represents the IS150 element's transposase. In vivo, the wild-type IS150 element generates circular excision products and linear IS150 molecules. Circular and linear species have previously been detected with mutant derivatives of other members of the IS3 family. Our finding supports the assumption that these products represent true transposition intermediates of members of this family. Analysis of the molecular nature of these two species suggested that the circular forms are precursors of the linear molecules. Elimination of InsA synthesis within the otherwise intact element led to accumulation of large amounts of the linear species, indicating that the primary role of InsA may be to prevent abortive production of the linear species and to couple generation of these species to productive insertion events.

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“…In addition, linear IS molecules accumulated in the case of IS150 (Fig. 2), IS3 (24,26), and IS911 (30).…”

Section: Discussionmentioning

confidence: 99%

Escherichia coliInsertion Sequence IS150: Transposition via Circular and Linear Intermediates

Haas

Rak

2002

J Bacteriol

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“…First, in both of these cases, the size of the intervening sequence between the IS ends (3 bp) was identical to the size of the target duplication associated with conventional transposition events. This coincidence spurred the suggestion that minicircles were formed by a process akin to 'reversal' of transposition -excision of the IS (using double-strand cuts with the same stagger as was made in the target during integration), followed by ligation of the IS ends into a circle (for example, see Sekine et al, 1996). The lack of coincidence between the IS2 target duplication (5 bp) and its minicircle junction (a 1 bp intervening sequence) suggest that such a scenario is unlikely to be correct.…”

Section: Is 2 Minicirclesmentioning

confidence: 99%

“…Subsequently, Sekine et al (1996) detected linear forms of IS3 and proposed that IS3 followed the Tn10/Tn7 paradigm, inferring that the minicircles were formed from the IS3 linears by ligation of the two ends. However, Polard and Chandler (1995b) detected, in high yield, a different product of IS911 transposition that they called a figure-eight.…”

Section: Fig 1 Derivatives Of Is2mentioning

confidence: 99%

Two abundant intramolecular transposition products, resulting from reactions initiated at a single end, suggest that IS2 transposes by an unconventional pathway

Lewis

Grindley

1997

Molecular Microbiology

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SummaryThe Escherichia coli insertion sequence, IS2, is a member of the IS3 family of bacterial transposable elements. Its transposase is a fusion protein, OrfAB, made by a programmed ¹1 translational frameshift near to the end of orfA and just after the start of orfB. We have characterized two major products of IS2 intramolecular transposition, which accumulate in cells that express the IS2 OrfAB fusion protein at elevated levels. The more abundant product is a minicircle composed of the complete IS2 with just a single basepair (occasionally 2 bp) separating the two IS ends. In all cases, this basepair is derived from the vector sequence immediately adjacent to the left IS2 end (IRL). The second product is a figure-eight molecule that contains all the IS2 and vector sequences present in the parental plasmid. One DNA strand contains the parental sequences unrearranged. The other contains a single-stranded version of the minicircle junction -the precise 3Ј end of IRR has been cleaved and joined to a target just outside the 5Ј end of IRL; the remaining vector sequences have a free 5Ј end, derived from cleavage at the 3Ј end of IRR, and a free 3Ј end, released upon cleavage of the target site adjacent to IRL. We propose that figure-eight molecules are the precursor to IS2 minicircles and that the formation of these two products is the initial step in IS2 intermolecular transposition. This proposed transposition pathway provides a means for a transposase that can cleave only one strand at each IS end to produce simple insertions and avoid forming co-integrates.

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“…ISY100, however, does not appear to be inserted into the palindromic sequences. Note that, unlike ISY100, some other prokaryotic transposable elements generate a linear DNA molecule with the 5Ј overhang or blunt ends as a transposition intermediate (2,3,31). Linear molecules generated from all of the elements, including ISY100, however, have the 3Ј ends coincident with the exact ends of the elements.…”

Section: Discussionmentioning

confidence: 99%

Transposition of Cyanobacterium Insertion Element ISY100inEscherichia coli

2002

Self Cite

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The genome of the cyanobacterium Synechocystis sp. strain PCC6803 has nine kinds of insertion sequence (IS) elements, of which ISY100 in 22 copies is the most abundant. A typical ISY100 member is 947 bp long and has imperfect terminal inverted repeat sequences. It has an open reading frame encoding a 282-amino-acid protein that appears to have partial homology with the transposase encoded by a bacterial IS, IS630, indicating that ISY100 belongs to the IS630 family. To determine whether ISY100 has transposition ability, we constructed a plasmid carrying the IPTG (isopropyl-␤-D-thiogalactopyranoside)-inducible transposase gene at one site and mini-ISY100 with the chloramphenicol resistance gene, substituted for the transposase gene of ISY100, at another site and introduced the plasmid into an Escherichia coli strain already harboring a target plasmid. Mini-ISY100 transposed to the target plasmid in the presence of IPTG at a very high frequency. Mini-ISY100 was inserted into the TA sequence and duplicated it upon transposition, as do IS630 family elements. Moreover, the mini-ISY100-carrying plasmid produced linear molecules of mini-ISY100 with the exact 3 ends of ISY100 and 5 ends lacking two nucleotides of the ISY100 sequence. No bacterial insertion elements have been shown to generate such molecules, whereas the eukaryotic Tc1/mariner family elements, Tc1 and Tc3, which transpose to the TA sequence, have. These findings suggest that ISY100 transposes to a new site through the formation of linear molecules, such as Tc1 and Tc3, by excision. Some Tc1/mariner family elements leave a footprint with an extra sequence at the site of excision. No footprints, however, were detected in the case of ISY100, suggesting that eukaryotes have a system that repairs a double strand break at the site of excision by an end-joining reaction, in which the gap is filled with a sequence of several base pairs, whereas prokaryotes do not have such a system. ISY100 transposes in E. coli, indicating that it transposes without any host factor other than the transposase encoded by itself. Therefore, it may be able to transpose in other biological systems.Transposable DNA elements that can move from one site to another are present in both prokaryotes and eukaryotes. These elements generally have terminal inverted repeat sequences (IRs) and a gene encoding transposase (for reviews, see references 18 and 22). Defective elements with a deletion in the transposase gene cannot transpose by themselves but can when transposase is supplied from another source. Upon transposition, they generate duplication of a sequence of several base pairs at the target site.The insertion sequence (IS) is a small transposable element in bacteria. IS elements are classified as several families based on the homology in the amino acid sequences of the transposases encoded by the elements, IRs, and the length of the target site sequence (see reference 18). Transposases usually have a DNA-binding domain with a helix-turn-helix structure and a catalytic domain with a DDE ...

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Identification and Characterization of the Linear IS3 Molecules Generated by Staggered Breaks

Cited by 21 publications

References 31 publications

Escherichia coliInsertion Sequence IS150: Transposition via Circular and Linear Intermediates

Escherichia coliInsertion Sequence IS150: Transposition via Circular and Linear Intermediates

Two abundant intramolecular transposition products, resulting from reactions initiated at a single end, suggest that IS2 transposes by an unconventional pathway

Transposition of Cyanobacterium Insertion Element ISY100inEscherichia coli

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