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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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Binding of full-length transposase to ISY100 IRR. A. A 58 bp DNA fragment containing 30 bp from the right end of ISY100 and 28 bp of flanking sequences (IRR58) was incubated with purified transposase, and complexes were separated by non-denaturing polyacrylamide gel electrophoresis. Unbound DNA (IRR58) and two complexes (I and II) are indicated. A twofold dilution series of transposase was used: Lane 1, no protein; lane 2, ∼3 nM transposase; lane 7, ∼100 nM transposase. B. Competition experiment. All lanes contain labelled IRR58 and 100 nM transposase. Lane 2, 3 and 4 contain 1 μM, 0.33 μM and 0.1 μM, respectively, of unlabelled IRL58 as competitor, whereas lanes 5, 6 and 7 contain the same concentrations of an unrelated 53 bp sequence.
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fig02: Binding of full-length transposase to ISY100 IRR. A. A 58 bp DNA fragment containing 30 bp from the right end of ISY100 and 28 bp of flanking sequences (IRR58) was incubated with purified transposase, and complexes were separated by non-denaturing polyacrylamide gel electrophoresis. Unbound DNA (IRR58) and two complexes (I and II) are indicated. A twofold dilution series of transposase was used: Lane 1, no protein; lane 2, ∼3 nM transposase; lane 7, ∼100 nM transposase. B. Competition experiment. All lanes contain labelled IRR58 and 100 nM transposase. Lane 2, 3 and 4 contain 1 μM, 0.33 μM and 0.1 μM, respectively, of unlabelled IRL58 as competitor, whereas lanes 5, 6 and 7 contain the same concentrations of an unrelated 53 bp sequence.

Mentions: To test whether transposase can bind to DNA, an electrophoretic mobility shift assay (EMSA) was carried out with purified transposase and a 58 bp labelled DNA fragment (IRR58) containing 30 bp from the right end of ISY100 together with 28 bp of flanking sequences. Transposase bound to IRR58 in the presence of an excess of poly(dI-dC) competitor, giving one major retarded complex (complex I, Fig. 2A). Transposase also bound to ISY100 IRL with a similar affinity (data not shown). Binding was abolished by the addition of excess unlabelled IRL58 competitor DNA, but not by the addition of an equivalent amount of unrelated DNA (Fig. 2B). Therefore the binding is specific for transposon inverted repeat sequences. On some gels, a minor slower migrating complex was observed (complex II; Fig. 2A). Addition of an excess of a larger (78 bp) unlabelled fragment containing IRL sequences (IRL78) reduced the mobility of the labelled complex II formed by IRR58, but had no effect on complex I (data not shown), demonstrating that complex I contains only one DNA fragment, while complex II contains at least two DNA fragments and may represent a paired end complex (PEC).


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

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

Binding of full-length transposase to ISY100 IRR. A. A 58 bp DNA fragment containing 30 bp from the right end of ISY100 and 28 bp of flanking sequences (IRR58) was incubated with purified transposase, and complexes were separated by non-denaturing polyacrylamide gel electrophoresis. Unbound DNA (IRR58) and two complexes (I and II) are indicated. A twofold dilution series of transposase was used: Lane 1, no protein; lane 2, ∼3 nM transposase; lane 7, ∼100 nM transposase. B. Competition experiment. All lanes contain labelled IRR58 and 100 nM transposase. Lane 2, 3 and 4 contain 1 μM, 0.33 μM and 0.1 μM, respectively, of unlabelled IRL58 as competitor, whereas lanes 5, 6 and 7 contain the same concentrations of an unrelated 53 bp sequence.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2170065&req=5

fig02: Binding of full-length transposase to ISY100 IRR. A. A 58 bp DNA fragment containing 30 bp from the right end of ISY100 and 28 bp of flanking sequences (IRR58) was incubated with purified transposase, and complexes were separated by non-denaturing polyacrylamide gel electrophoresis. Unbound DNA (IRR58) and two complexes (I and II) are indicated. A twofold dilution series of transposase was used: Lane 1, no protein; lane 2, ∼3 nM transposase; lane 7, ∼100 nM transposase. B. Competition experiment. All lanes contain labelled IRR58 and 100 nM transposase. Lane 2, 3 and 4 contain 1 μM, 0.33 μM and 0.1 μM, respectively, of unlabelled IRL58 as competitor, whereas lanes 5, 6 and 7 contain the same concentrations of an unrelated 53 bp sequence.
Mentions: To test whether transposase can bind to DNA, an electrophoretic mobility shift assay (EMSA) was carried out with purified transposase and a 58 bp labelled DNA fragment (IRR58) containing 30 bp from the right end of ISY100 together with 28 bp of flanking sequences. Transposase bound to IRR58 in the presence of an excess of poly(dI-dC) competitor, giving one major retarded complex (complex I, Fig. 2A). Transposase also bound to ISY100 IRL with a similar affinity (data not shown). Binding was abolished by the addition of excess unlabelled IRL58 competitor DNA, but not by the addition of an equivalent amount of unrelated DNA (Fig. 2B). Therefore the binding is specific for transposon inverted repeat sequences. On some gels, a minor slower migrating complex was observed (complex II; Fig. 2A). Addition of an excess of a larger (78 bp) unlabelled fragment containing IRL sequences (IRL78) reduced the mobility of the labelled complex II formed by IRR58, but had no effect on complex I (data not shown), demonstrating that complex I contains only one DNA fragment, while complex II contains at least two DNA fragments and may represent a paired end complex (PEC).

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