Limits...
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.

Show MeSH

Related in: MedlinePlus

In vitro excision and transposition of ISY100. A. Supercoiled pISY100-kan (4.0 kb), containing a 1.3 kb kanamycin resistance fragment flanked by IRL and IRR in pUC18, was incubated for 16 h in the presence of transposase (100 nM) and divalent cations as indicated. Products were separated by agarose gel electrophoresis. Bands are indicated as follows: sc, lin and oc, supercoiled, linear and open circle pISY100-kan, respectively; 2mer lin and 2mer oc, linear and open circle dimer; ETF, excised mini-transposon fragment; vector, vector backbone. Proposed intermolecular transposition products (σ1 and σ2) are indicated schematically. B. Supercoiled pUC4K target plasmid (3.9 kb) was incubated with or without purified excised mini-transposon fragment and transposase as indicated. Bands are indicated as in (A) except for supercoiled and open circle pUC4K, sc target and oc target. C. Proposed transposition pathway. Single-strand cleavage at one or both transposon ends produces open circle. Cleavage of the other strand at one or both ends releases ETF, vector and linearized substrate. Linear and ETF fragments can transpose into another copy of the circular substrate to produce the intermolecular transposition products shown.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2170065&req=5

fig05: In vitro excision and transposition of ISY100. A. Supercoiled pISY100-kan (4.0 kb), containing a 1.3 kb kanamycin resistance fragment flanked by IRL and IRR in pUC18, was incubated for 16 h in the presence of transposase (100 nM) and divalent cations as indicated. Products were separated by agarose gel electrophoresis. Bands are indicated as follows: sc, lin and oc, supercoiled, linear and open circle pISY100-kan, respectively; 2mer lin and 2mer oc, linear and open circle dimer; ETF, excised mini-transposon fragment; vector, vector backbone. Proposed intermolecular transposition products (σ1 and σ2) are indicated schematically. B. Supercoiled pUC4K target plasmid (3.9 kb) was incubated with or without purified excised mini-transposon fragment and transposase as indicated. Bands are indicated as in (A) except for supercoiled and open circle pUC4K, sc target and oc target. C. Proposed transposition pathway. Single-strand cleavage at one or both transposon ends produces open circle. Cleavage of the other strand at one or both ends releases ETF, vector and linearized substrate. Linear and ETF fragments can transpose into another copy of the circular substrate to produce the intermolecular transposition products shown.

Mentions: To test whether ISY100 transposase has catalytic activity, a plasmid (pISY100-kan) containing an artificial mini-transposon, consisting of IRL and IRR flanking a kanamycin-resistance gene, was incubated in a simple buffer with purified full-length transposase (Fig. 5A). Supercoiled pISY100-kan was converted to a number of different products in a reaction that was dependent on transposase and either Mg2+ or Mn2+ divalent cations. The reaction produced linearized plasmid and two fragments of the correct size to be excised mini-transposon (ETF) and the vector backbone. Restriction digestion confirmed that these products were produced by double-strand cleavage at IRR and IRL. Most of the linearized plasmid was produced by cleavage at IRL (∼80%) while the rest (∼20%) was produced by cleavage at IRR (data not shown). A significant amount of nicked (open circle) plasmid was also produced by transposase in these reactions (Fig. 5A, lanes 3 and 4). It seems likely that transposase has cleaved one strand at one or both transposon ends in these open circular products. Nicked and linear dimer, produced from circular dimer present in the substrate, were also observed (Fig. 5A, lanes 3 and 4).


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

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

In vitro excision and transposition of ISY100. A. Supercoiled pISY100-kan (4.0 kb), containing a 1.3 kb kanamycin resistance fragment flanked by IRL and IRR in pUC18, was incubated for 16 h in the presence of transposase (100 nM) and divalent cations as indicated. Products were separated by agarose gel electrophoresis. Bands are indicated as follows: sc, lin and oc, supercoiled, linear and open circle pISY100-kan, respectively; 2mer lin and 2mer oc, linear and open circle dimer; ETF, excised mini-transposon fragment; vector, vector backbone. Proposed intermolecular transposition products (σ1 and σ2) are indicated schematically. B. Supercoiled pUC4K target plasmid (3.9 kb) was incubated with or without purified excised mini-transposon fragment and transposase as indicated. Bands are indicated as in (A) except for supercoiled and open circle pUC4K, sc target and oc target. C. Proposed transposition pathway. Single-strand cleavage at one or both transposon ends produces open circle. Cleavage of the other strand at one or both ends releases ETF, vector and linearized substrate. Linear and ETF fragments can transpose into another copy of the circular substrate to produce the intermolecular transposition products shown.
© Copyright Policy
Related In: Results  -  Collection

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

fig05: In vitro excision and transposition of ISY100. A. Supercoiled pISY100-kan (4.0 kb), containing a 1.3 kb kanamycin resistance fragment flanked by IRL and IRR in pUC18, was incubated for 16 h in the presence of transposase (100 nM) and divalent cations as indicated. Products were separated by agarose gel electrophoresis. Bands are indicated as follows: sc, lin and oc, supercoiled, linear and open circle pISY100-kan, respectively; 2mer lin and 2mer oc, linear and open circle dimer; ETF, excised mini-transposon fragment; vector, vector backbone. Proposed intermolecular transposition products (σ1 and σ2) are indicated schematically. B. Supercoiled pUC4K target plasmid (3.9 kb) was incubated with or without purified excised mini-transposon fragment and transposase as indicated. Bands are indicated as in (A) except for supercoiled and open circle pUC4K, sc target and oc target. C. Proposed transposition pathway. Single-strand cleavage at one or both transposon ends produces open circle. Cleavage of the other strand at one or both ends releases ETF, vector and linearized substrate. Linear and ETF fragments can transpose into another copy of the circular substrate to produce the intermolecular transposition products shown.
Mentions: To test whether ISY100 transposase has catalytic activity, a plasmid (pISY100-kan) containing an artificial mini-transposon, consisting of IRL and IRR flanking a kanamycin-resistance gene, was incubated in a simple buffer with purified full-length transposase (Fig. 5A). Supercoiled pISY100-kan was converted to a number of different products in a reaction that was dependent on transposase and either Mg2+ or Mn2+ divalent cations. The reaction produced linearized plasmid and two fragments of the correct size to be excised mini-transposon (ETF) and the vector backbone. Restriction digestion confirmed that these products were produced by double-strand cleavage at IRR and IRL. Most of the linearized plasmid was produced by cleavage at IRL (∼80%) while the rest (∼20%) was produced by cleavage at IRR (data not shown). A significant amount of nicked (open circle) plasmid was also produced by transposase in these reactions (Fig. 5A, lanes 3 and 4). It seems likely that transposase has cleaved one strand at one or both transposon ends in these open circular products. Nicked and linear dimer, produced from circular dimer present in the substrate, were also observed (Fig. 5A, lanes 3 and 4).

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