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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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Mapping the cleavage sites on ISY100 ends. A. IRL58 was 5′ end-labelled with 32P on the top strand and incubated in the presence of 20% DMSO with or without transposase as indicated. Reactions were run on an 8% polyacrylamide sequencing gel, alongside a marker ladder produced by limited DNase I cleavage of the same DNA. Labelled single-stranded oligonucleotides (T+2 and T−1), representing predicted cleavage products, were run as additional markers. B. IRL58 was 5′ end-labelled with 32P on the bottom strand and treated as in A. Labelled single-stranded oligonucleotide representing the predicted cleavage product (B0) was run as marker. C. IRR58 was 5′ end-labelled with 32P on the top strand and incubated with transposase in the presence or absence of 20% DMSO as indicated. Reactions were run on an 8% polyacrylamide sequencing gel adjacent to a DNase I ladder produced from the same labelled DNA. Additional markers were produced by cleavage with AcuI or AluI. D. IRR58 was 5′ end-labelled with 32P on the bottom strand and treated as in C. Major and minor transposase cleavage sites (large and small arrows), and marker sizes are indicated on the DNA sequence below the gels.
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fig06: Mapping the cleavage sites on ISY100 ends. A. IRL58 was 5′ end-labelled with 32P on the top strand and incubated in the presence of 20% DMSO with or without transposase as indicated. Reactions were run on an 8% polyacrylamide sequencing gel, alongside a marker ladder produced by limited DNase I cleavage of the same DNA. Labelled single-stranded oligonucleotides (T+2 and T−1), representing predicted cleavage products, were run as additional markers. B. IRL58 was 5′ end-labelled with 32P on the bottom strand and treated as in A. Labelled single-stranded oligonucleotide representing the predicted cleavage product (B0) was run as marker. C. IRR58 was 5′ end-labelled with 32P on the top strand and incubated with transposase in the presence or absence of 20% DMSO as indicated. Reactions were run on an 8% polyacrylamide sequencing gel adjacent to a DNase I ladder produced from the same labelled DNA. Additional markers were produced by cleavage with AcuI or AluI. D. IRR58 was 5′ end-labelled with 32P on the bottom strand and treated as in C. Major and minor transposase cleavage sites (large and small arrows), and marker sizes are indicated on the DNA sequence below the gels.

Mentions: To map the exact sites of cleavage at ISY100 ends, double-stranded oligonucleotides containing IRR or IRL sequences were 5′ end-labelled on the top or bottom strands, and incubated with transposase in the presence of Mg2+ and DMSO. Reaction products were run on a strand-separating polyacrylamide gel adjacent to appropriate single-stranded markers. Top strand cleavage (yielding a transposon 5′ end) occurred at several positions close to the transposon end. A strong cut site was present two nucleotides inside the transposon on both IRR and IRL, but cleavage also occurred one to eight nucleotides outside the transposon at both ends (Fig. 6A and C). On the bottom strand, the majority of cleavage was one nucleotide inside the transposon, although a small amount of cleavage was detected precisely at the transposon 3′ end (Fig. 6B and D).


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

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

Mapping the cleavage sites on ISY100 ends. A. IRL58 was 5′ end-labelled with 32P on the top strand and incubated in the presence of 20% DMSO with or without transposase as indicated. Reactions were run on an 8% polyacrylamide sequencing gel, alongside a marker ladder produced by limited DNase I cleavage of the same DNA. Labelled single-stranded oligonucleotides (T+2 and T−1), representing predicted cleavage products, were run as additional markers. B. IRL58 was 5′ end-labelled with 32P on the bottom strand and treated as in A. Labelled single-stranded oligonucleotide representing the predicted cleavage product (B0) was run as marker. C. IRR58 was 5′ end-labelled with 32P on the top strand and incubated with transposase in the presence or absence of 20% DMSO as indicated. Reactions were run on an 8% polyacrylamide sequencing gel adjacent to a DNase I ladder produced from the same labelled DNA. Additional markers were produced by cleavage with AcuI or AluI. D. IRR58 was 5′ end-labelled with 32P on the bottom strand and treated as in C. Major and minor transposase cleavage sites (large and small arrows), and marker sizes are indicated on the DNA sequence below the gels.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2170065&req=5

fig06: Mapping the cleavage sites on ISY100 ends. A. IRL58 was 5′ end-labelled with 32P on the top strand and incubated in the presence of 20% DMSO with or without transposase as indicated. Reactions were run on an 8% polyacrylamide sequencing gel, alongside a marker ladder produced by limited DNase I cleavage of the same DNA. Labelled single-stranded oligonucleotides (T+2 and T−1), representing predicted cleavage products, were run as additional markers. B. IRL58 was 5′ end-labelled with 32P on the bottom strand and treated as in A. Labelled single-stranded oligonucleotide representing the predicted cleavage product (B0) was run as marker. C. IRR58 was 5′ end-labelled with 32P on the top strand and incubated with transposase in the presence or absence of 20% DMSO as indicated. Reactions were run on an 8% polyacrylamide sequencing gel adjacent to a DNase I ladder produced from the same labelled DNA. Additional markers were produced by cleavage with AcuI or AluI. D. IRR58 was 5′ end-labelled with 32P on the bottom strand and treated as in C. Major and minor transposase cleavage sites (large and small arrows), and marker sizes are indicated on the DNA sequence below the gels.
Mentions: To map the exact sites of cleavage at ISY100 ends, double-stranded oligonucleotides containing IRR or IRL sequences were 5′ end-labelled on the top or bottom strands, and incubated with transposase in the presence of Mg2+ and DMSO. Reaction products were run on a strand-separating polyacrylamide gel adjacent to appropriate single-stranded markers. Top strand cleavage (yielding a transposon 5′ end) occurred at several positions close to the transposon end. A strong cut site was present two nucleotides inside the transposon on both IRR and IRL, but cleavage also occurred one to eight nucleotides outside the transposon at both ends (Fig. 6A and C). On the bottom strand, the majority of cleavage was one nucleotide inside the transposon, although a small amount of cleavage was detected precisely at the transposon 3′ end (Fig. 6B and D).

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