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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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DNase I footprint of full-length transposase bound to IRR. A. Transposase protects the inverted repeat sequence (shown as an arrow) on both strands of IRR. B. DNA sequence of the restriction fragment used in footprinting experiments. The 49 bp derived from the right end of ISY100 are shown in capital letters and the 24 bp inverted repeat is indicated with an arrow. The extent of protection from DNase I on top and bottom strands is indicated by black bars above and below the sequence respectively.
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fig03: DNase I footprint of full-length transposase bound to IRR. A. Transposase protects the inverted repeat sequence (shown as an arrow) on both strands of IRR. B. DNA sequence of the restriction fragment used in footprinting experiments. The 49 bp derived from the right end of ISY100 are shown in capital letters and the 24 bp inverted repeat is indicated with an arrow. The extent of protection from DNase I on top and bottom strands is indicated by black bars above and below the sequence respectively.

Mentions: To ascertain where transposase binds on the transposon ends, DNase I footprinting experiments were carried out on DNA fragments containing ISY100 IRR (Fig. 3A). Almost all of the 24 bp IRR sequence was protected on the top strand, while the complete IRR sequence and a few nucleotides on either side were protected on the bottom strand (Fig. 3B). A similar pattern of protection was observed on DNA fragments containing IRL (data not shown).


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

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

DNase I footprint of full-length transposase bound to IRR. A. Transposase protects the inverted repeat sequence (shown as an arrow) on both strands of IRR. B. DNA sequence of the restriction fragment used in footprinting experiments. The 49 bp derived from the right end of ISY100 are shown in capital letters and the 24 bp inverted repeat is indicated with an arrow. The extent of protection from DNase I on top and bottom strands is indicated by black bars above and below the sequence respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2170065&req=5

fig03: DNase I footprint of full-length transposase bound to IRR. A. Transposase protects the inverted repeat sequence (shown as an arrow) on both strands of IRR. B. DNA sequence of the restriction fragment used in footprinting experiments. The 49 bp derived from the right end of ISY100 are shown in capital letters and the 24 bp inverted repeat is indicated with an arrow. The extent of protection from DNase I on top and bottom strands is indicated by black bars above and below the sequence respectively.
Mentions: To ascertain where transposase binds on the transposon ends, DNase I footprinting experiments were carried out on DNA fragments containing ISY100 IRR (Fig. 3A). Almost all of the 24 bp IRR sequence was protected on the top strand, while the complete IRR sequence and a few nucleotides on either side were protected on the bottom strand (Fig. 3B). A similar pattern of protection was observed on DNA fragments containing IRL (data not shown).

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