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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 N-terminal transposase derivatives to the ISY100 inverted repeat. A. Transposase deletion derivatives were purified by metal affinity chromatography and analysed by Coomassie-stained Tris-tricine SDS-PAGE. B. The indicated protein (1.25 μM) was incubated with IRL58, and protein–DNA complexes were separated by non-denaturing polyacrylamide gel electrophoresis. C. The indicated concentrations of Tnp1−77 and Tnp1−95 were incubated with IRL58 and complexes were separated as in (B).
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fig04: Binding of N-terminal transposase derivatives to the ISY100 inverted repeat. A. Transposase deletion derivatives were purified by metal affinity chromatography and analysed by Coomassie-stained Tris-tricine SDS-PAGE. B. The indicated protein (1.25 μM) was incubated with IRL58, and protein–DNA complexes were separated by non-denaturing polyacrylamide gel electrophoresis. C. The indicated concentrations of Tnp1−77 and Tnp1−95 were incubated with IRL58 and complexes were separated as in (B).

Mentions: N-terminal DNA-binding domains that specifically recognize their transposon terminal sequences have been identified in a number of Tc1/mariner family transposases (Colloms et al., 1994; Vos and Plasterk, 1994; Auge-Gouillou et al., 2001; Zhang et al., 2001; Izsvak et al., 2002; Watkins et al., 2004; Feschotte et al., 2005). To test whether ISY100 transposase contains a similar DNA-binding domain, N-terminal derivatives containing the first 37, 38, 46, 57, 68, 77, 95 or 110 amino acids of ISY100 transposase (Tnp1−37 … Tnp1−110) were expressed in E. coli and tested for their ability to bind to DNA. Crude extracts from E. coli expressing Tnp1−37, Tnp1−38 and Tnp1−45 failed to bind to ISY100 inverted repeat sequences, while crude extracts containing Tnp1−57, Tnp1−68, Tnp1−77, Tnp1−95 and Tnp1−110 did bind (data not shown). To investigate this further, Tnp1−57, Tnp1−68, Tnp1−77, Tnp1−95 and Tnp1−110 were purified by nickel affinity chromatography (Fig. 4A) and used in EMSAs with IRL58. Tnp1−57, Tnp1−68, Tnp1−77 bound to IRL58, giving a series of three to four retarded complexes that decreased in mobility with increasing protein length (Fig. 4B). Tnp1−95 and Tnp1−110 also produced a series of retarded complexes with IRL58, but there was a step shift in behaviour between Tnp1−77 and Tnp1−95 (Fig. 4B). The fastest-migrating complexes produced by Tnp1−95 and Tnp1−110 had higher mobilities than the fastest complex produced by Tnp1−77. To see if this step shift was reflected in the binding affinity of these two proteins, dilutions of Tnp1−77 and Tnp1−95 were used in EMSAs with IRL58 (Fig. 4C). Both proteins gave a series of retarded bands, with the slower-migrating complexes appearing only as the protein concentration was increased above approximately 100 nM. Experiments with mixtures of different-length DNA fragments gave no indication that any of these complexes contained more than one DNA fragment (data not shown). The slower-migrating complexes most likely result from binding of multiple protein subunits to IRL58, either by non-specific DNA binding or by protein–protein interactions with specifically bound subunits. Tnp1−95 bound to IRL58 about 16-fold more tightly than Tnp1−77, with apparent disassociation constants (Kd) for the first complexes of approximately 10 nM and 160 nM respectively. This difference in affinity was also reflected in DNase I footprinting experiments on transposon ends. Tnp1−95 gave almost identical protection to the full-length transposase, whereas no specific protection was observed with Tnp1−57 and Tnp1−77 (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)

Binding of N-terminal transposase derivatives to the ISY100 inverted repeat. A. Transposase deletion derivatives were purified by metal affinity chromatography and analysed by Coomassie-stained Tris-tricine SDS-PAGE. B. The indicated protein (1.25 μM) was incubated with IRL58, and protein–DNA complexes were separated by non-denaturing polyacrylamide gel electrophoresis. C. The indicated concentrations of Tnp1−77 and Tnp1−95 were incubated with IRL58 and complexes were separated as in (B).
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Related In: Results  -  Collection

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fig04: Binding of N-terminal transposase derivatives to the ISY100 inverted repeat. A. Transposase deletion derivatives were purified by metal affinity chromatography and analysed by Coomassie-stained Tris-tricine SDS-PAGE. B. The indicated protein (1.25 μM) was incubated with IRL58, and protein–DNA complexes were separated by non-denaturing polyacrylamide gel electrophoresis. C. The indicated concentrations of Tnp1−77 and Tnp1−95 were incubated with IRL58 and complexes were separated as in (B).
Mentions: N-terminal DNA-binding domains that specifically recognize their transposon terminal sequences have been identified in a number of Tc1/mariner family transposases (Colloms et al., 1994; Vos and Plasterk, 1994; Auge-Gouillou et al., 2001; Zhang et al., 2001; Izsvak et al., 2002; Watkins et al., 2004; Feschotte et al., 2005). To test whether ISY100 transposase contains a similar DNA-binding domain, N-terminal derivatives containing the first 37, 38, 46, 57, 68, 77, 95 or 110 amino acids of ISY100 transposase (Tnp1−37 … Tnp1−110) were expressed in E. coli and tested for their ability to bind to DNA. Crude extracts from E. coli expressing Tnp1−37, Tnp1−38 and Tnp1−45 failed to bind to ISY100 inverted repeat sequences, while crude extracts containing Tnp1−57, Tnp1−68, Tnp1−77, Tnp1−95 and Tnp1−110 did bind (data not shown). To investigate this further, Tnp1−57, Tnp1−68, Tnp1−77, Tnp1−95 and Tnp1−110 were purified by nickel affinity chromatography (Fig. 4A) and used in EMSAs with IRL58. Tnp1−57, Tnp1−68, Tnp1−77 bound to IRL58, giving a series of three to four retarded complexes that decreased in mobility with increasing protein length (Fig. 4B). Tnp1−95 and Tnp1−110 also produced a series of retarded complexes with IRL58, but there was a step shift in behaviour between Tnp1−77 and Tnp1−95 (Fig. 4B). The fastest-migrating complexes produced by Tnp1−95 and Tnp1−110 had higher mobilities than the fastest complex produced by Tnp1−77. To see if this step shift was reflected in the binding affinity of these two proteins, dilutions of Tnp1−77 and Tnp1−95 were used in EMSAs with IRL58 (Fig. 4C). Both proteins gave a series of retarded bands, with the slower-migrating complexes appearing only as the protein concentration was increased above approximately 100 nM. Experiments with mixtures of different-length DNA fragments gave no indication that any of these complexes contained more than one DNA fragment (data not shown). The slower-migrating complexes most likely result from binding of multiple protein subunits to IRL58, either by non-specific DNA binding or by protein–protein interactions with specifically bound subunits. Tnp1−95 bound to IRL58 about 16-fold more tightly than Tnp1−77, with apparent disassociation constants (Kd) for the first complexes of approximately 10 nM and 160 nM respectively. This difference in affinity was also reflected in DNase I footprinting experiments on transposon ends. Tnp1−95 gave almost identical protection to the full-length transposase, whereas no specific protection was observed with Tnp1−57 and Tnp1−77 (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