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In vitro transposition of ISY100, a bacterial insertion sequence belonging to the Tc1/mariner family.

Feng X, Colloms SD - Mol. Microbiol. (2007)

Bottom Line: Transposase made double-strand breaks on a supercoiled DNA molecule containing a mini-ISY100 transposon, cleaving exactly at the transposon 3' ends and two nucleotides inside the 5' ends.Cleavage of short linear substrates containing a single transposon end was less precise.Transposase also catalysed strand transfer, covalently joining the transposon 3' end to the target DNA.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedical and Life Sciences, Division of Molecular Genetics, University of Glasgow, Anderson College, 56 Dumbarton Rd, Glasgow G11 6NU, Scotland, UK.

ABSTRACT
The Synechocystis sp. PCC6803 insertion sequence ISY100 (ISTcSa) belongs to the Tc1/mariner/IS630 family of transposable elements. ISY100 transposase was purified and shown to promote transposition in vitro. Transposase binds specifically to ISY100 terminal inverted repeat sequences via an N-terminal DNA-binding domain containing two helix-turn-helix motifs. Transposase is the only protein required for excision and integration of ISY100. Transposase made double-strand breaks on a supercoiled DNA molecule containing a mini-ISY100 transposon, cleaving exactly at the transposon 3' ends and two nucleotides inside the 5' ends. Cleavage of short linear substrates containing a single transposon end was less precise. Transposase also catalysed strand transfer, covalently joining the transposon 3' end to the target DNA. When a donor plasmid carrying a mini-ISY100 was incubated with a target plasmid and transposase, the most common products were insertions of one transposon end into the target DNA, but insertions of both ends at a single target site could be recovered after transformation into Escherichia coli. Insertions were almost exclusively into TA dinucleotides, and the target TA was duplicated on insertion. Our results demonstrate that there are no fundamental differences between the transposition mechanisms of IS630 family elements in bacteria and Tc1/mariner elements in higher eukaryotes.

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The structure of ISY100. A. ISY100 encodes a single protein (transposase) flanked by two imperfect inverted repeats (IRL and IRR). Transposase contains two N-terminal helix–turn–helix DNA-binding motifs (HTH), and a C-terminal domain with homology to the DDE superfamily of DNA transposases. B. The sequences of IRL and IRR are shown aligned. Vertical lines show positions of identity and the flanking TA target duplication is shown lower case and boxed. The orientation of the inverted repeat sequences is indicated by an arrow above the sequences as in (A). C. Comparison of ISY100 with other members of the Tc1/mariner family. Transposase lengths are shown in amino acids (aa). The phylogenetic tree was produced by PHYLIP NEIGHBOUR from a MUSCLE alignment of the C-terminal domains as shown in Fig. S2 (SB, Sleeping Beauty). Nodes supported by bootstrap values > 95% are indicated (*).
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fig01: The structure of ISY100. A. ISY100 encodes a single protein (transposase) flanked by two imperfect inverted repeats (IRL and IRR). Transposase contains two N-terminal helix–turn–helix DNA-binding motifs (HTH), and a C-terminal domain with homology to the DDE superfamily of DNA transposases. B. The sequences of IRL and IRR are shown aligned. Vertical lines show positions of identity and the flanking TA target duplication is shown lower case and boxed. The orientation of the inverted repeat sequences is indicated by an arrow above the sequences as in (A). C. Comparison of ISY100 with other members of the Tc1/mariner family. Transposase lengths are shown in amino acids (aa). The phylogenetic tree was produced by PHYLIP NEIGHBOUR from a MUSCLE alignment of the C-terminal domains as shown in Fig. S2 (SB, Sleeping Beauty). Nodes supported by bootstrap values > 95% are indicated (*).

Mentions: Members of the IS630 family of Insertion Sequences are found in a wide range of bacterial species, from Proteobacteria such as Escherichia coli O157 to Cyanobacteria and Archaebacteria (Siguier et al., 2006). These elements appear to be distantly related to the Tc1/mariner family of transposons. They have a similar structure: they are flanked by apparent TA target duplications and carry single transposase genes whose products share sequence similarity with Tc1/mariner transposases (Fig. 1; Mahillon and Chandler, 1998). Two members of the IS630 family, IS630 from Shigella sonnei (Tenzen et al., 1990) and ISY100 (otherwise known as ISS1987 and ISTcSa) from Synechocystis sp. PCC6803 (Cassier-Chauvat et al., 1997; Urasaki et al., 2002), have been studied in vivo. Both elements transpose in E. coli upon expression of their cognate transposase protein. They are excised by double-strand breaks at the transposon ends and insert into TA dinucleotides with characteristic two base-pair target duplications (Tenzen et al., 1990; Tenzen and Ohtsubo, 1991; Urasaki et al., 2002). However, to date no bacterial elements from this family have been studied in vitro. To study the mechanism of transposition of these bacterial elements in more detail, and to allow comparison with their eukaryotic counterparts, we have set up an in vitro transposition system for ISY100. Here we report that transposase is the sole protein required for transposition and demonstrate that transposition occurs by a cut and paste mechanism similar to that of other characterized members of the Tc1/mariner family. Our results demonstrate the evolutionary conservation of transposition mechanism of Tc1/mariner/IS630 elements, and provide a genetically and biochemically tractable system for the study of this family.


In vitro transposition of ISY100, a bacterial insertion sequence belonging to the Tc1/mariner family.

Feng X, Colloms SD - Mol. Microbiol. (2007)

The structure of ISY100. A. ISY100 encodes a single protein (transposase) flanked by two imperfect inverted repeats (IRL and IRR). Transposase contains two N-terminal helix–turn–helix DNA-binding motifs (HTH), and a C-terminal domain with homology to the DDE superfamily of DNA transposases. B. The sequences of IRL and IRR are shown aligned. Vertical lines show positions of identity and the flanking TA target duplication is shown lower case and boxed. The orientation of the inverted repeat sequences is indicated by an arrow above the sequences as in (A). C. Comparison of ISY100 with other members of the Tc1/mariner family. Transposase lengths are shown in amino acids (aa). The phylogenetic tree was produced by PHYLIP NEIGHBOUR from a MUSCLE alignment of the C-terminal domains as shown in Fig. S2 (SB, Sleeping Beauty). Nodes supported by bootstrap values > 95% are indicated (*).
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Related In: Results  -  Collection

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fig01: The structure of ISY100. A. ISY100 encodes a single protein (transposase) flanked by two imperfect inverted repeats (IRL and IRR). Transposase contains two N-terminal helix–turn–helix DNA-binding motifs (HTH), and a C-terminal domain with homology to the DDE superfamily of DNA transposases. B. The sequences of IRL and IRR are shown aligned. Vertical lines show positions of identity and the flanking TA target duplication is shown lower case and boxed. The orientation of the inverted repeat sequences is indicated by an arrow above the sequences as in (A). C. Comparison of ISY100 with other members of the Tc1/mariner family. Transposase lengths are shown in amino acids (aa). The phylogenetic tree was produced by PHYLIP NEIGHBOUR from a MUSCLE alignment of the C-terminal domains as shown in Fig. S2 (SB, Sleeping Beauty). Nodes supported by bootstrap values > 95% are indicated (*).
Mentions: Members of the IS630 family of Insertion Sequences are found in a wide range of bacterial species, from Proteobacteria such as Escherichia coli O157 to Cyanobacteria and Archaebacteria (Siguier et al., 2006). These elements appear to be distantly related to the Tc1/mariner family of transposons. They have a similar structure: they are flanked by apparent TA target duplications and carry single transposase genes whose products share sequence similarity with Tc1/mariner transposases (Fig. 1; Mahillon and Chandler, 1998). Two members of the IS630 family, IS630 from Shigella sonnei (Tenzen et al., 1990) and ISY100 (otherwise known as ISS1987 and ISTcSa) from Synechocystis sp. PCC6803 (Cassier-Chauvat et al., 1997; Urasaki et al., 2002), have been studied in vivo. Both elements transpose in E. coli upon expression of their cognate transposase protein. They are excised by double-strand breaks at the transposon ends and insert into TA dinucleotides with characteristic two base-pair target duplications (Tenzen et al., 1990; Tenzen and Ohtsubo, 1991; Urasaki et al., 2002). However, to date no bacterial elements from this family have been studied in vitro. To study the mechanism of transposition of these bacterial elements in more detail, and to allow comparison with their eukaryotic counterparts, we have set up an in vitro transposition system for ISY100. Here we report that transposase is the sole protein required for transposition and demonstrate that transposition occurs by a cut and paste mechanism similar to that of other characterized members of the Tc1/mariner family. Our results demonstrate the evolutionary conservation of transposition mechanism of Tc1/mariner/IS630 elements, and provide a genetically and biochemically tractable system for the study of this family.

Bottom Line: Transposase made double-strand breaks on a supercoiled DNA molecule containing a mini-ISY100 transposon, cleaving exactly at the transposon 3' ends and two nucleotides inside the 5' ends.Cleavage of short linear substrates containing a single transposon end was less precise.Transposase also catalysed strand transfer, covalently joining the transposon 3' end to the target DNA.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedical and Life Sciences, Division of Molecular Genetics, University of Glasgow, Anderson College, 56 Dumbarton Rd, Glasgow G11 6NU, Scotland, UK.

ABSTRACT
The Synechocystis sp. PCC6803 insertion sequence ISY100 (ISTcSa) belongs to the Tc1/mariner/IS630 family of transposable elements. ISY100 transposase was purified and shown to promote transposition in vitro. Transposase binds specifically to ISY100 terminal inverted repeat sequences via an N-terminal DNA-binding domain containing two helix-turn-helix motifs. Transposase is the only protein required for excision and integration of ISY100. Transposase made double-strand breaks on a supercoiled DNA molecule containing a mini-ISY100 transposon, cleaving exactly at the transposon 3' ends and two nucleotides inside the 5' ends. Cleavage of short linear substrates containing a single transposon end was less precise. Transposase also catalysed strand transfer, covalently joining the transposon 3' end to the target DNA. When a donor plasmid carrying a mini-ISY100 was incubated with a target plasmid and transposase, the most common products were insertions of one transposon end into the target DNA, but insertions of both ends at a single target site could be recovered after transformation into Escherichia coli. Insertions were almost exclusively into TA dinucleotides, and the target TA was duplicated on insertion. Our results demonstrate that there are no fundamental differences between the transposition mechanisms of IS630 family elements in bacteria and Tc1/mariner elements in higher eukaryotes.

Show MeSH
Related in: MedlinePlus