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

The 3′ end of ISY100 becomes covalently linked to the target DNA in the strand transfer reaction. A. A pre-cleaved transposon end, 5′ end-labelled on either the top or bottom strand, was incubated with transposase and a supercoiled target plasmid. Samples were withdrawn at the indicated times and run on a non-denaturing agarose gel (TAE) or a strand-separating alkaline gel (NaOH). DNA was detected by ethidium staining, and by autoradiography (32P). The positions of supercoiled (sc) and open circle (oc) target plasmid are shown to the left of the ethidium-stained gel. The positions of single end-joined (SEJ) and double end-joined (DEJ) strand transfer products are shown to the right of the autoradiographs. SEJ and DEJ products are indistinguishable on the strand separating gel. Unreacted labelled substrate has been cropped from the bottom of both autoradiographs. B. Sequence of the pre-cleaved end and schematic diagram of the reaction products.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2170065&req=5

fig08: The 3′ end of ISY100 becomes covalently linked to the target DNA in the strand transfer reaction. A. A pre-cleaved transposon end, 5′ end-labelled on either the top or bottom strand, was incubated with transposase and a supercoiled target plasmid. Samples were withdrawn at the indicated times and run on a non-denaturing agarose gel (TAE) or a strand-separating alkaline gel (NaOH). DNA was detected by ethidium staining, and by autoradiography (32P). The positions of supercoiled (sc) and open circle (oc) target plasmid are shown to the left of the ethidium-stained gel. The positions of single end-joined (SEJ) and double end-joined (DEJ) strand transfer products are shown to the right of the autoradiographs. SEJ and DEJ products are indistinguishable on the strand separating gel. Unreacted labelled substrate has been cropped from the bottom of both autoradiographs. B. Sequence of the pre-cleaved end and schematic diagram of the reaction products.

Mentions: To investigate the strand transfer activity of transposase, we used double-stranded oligonucleotides representing cleaved transposon ends. The termini were chosen to be the same as those produced on supercoiled plasmid substrates, exactly at the transposon 3′ end and two nucleotides inside the transposon at the 5′ end. Pre-cleaved transposon ends were labelled on either the top or bottom strand, and incubated together with a supercoiled target plasmid and transposase in a time course experiment. The products were run on a non-denaturing agarose gel and detected by autoradiography. Radioactive products co-migrating with the open circular and linear forms of the target plasmid were produced only in the presence of transposase, divalent metal ions and target plasmid (Fig. 8A and data not shown). The mobilities of these products are consistent with transfer of one or two transposon ends to a target plasmid, yielding nicked circle with one attached transposon end, and a linear molecule with two attached transposon ends respectively (Fig. 8B). Strand transfer products accumulated over time, and were observed irrespective of whether the top or the bottom strand was labelled (Fig. 8A). When the same reactions were run on a denaturing agarose gel, plasmid-sized labelled products were observed only when the transposon end was labelled on the bottom strand (Fig. 8A). Thus only the bottom strand, containing the transposon 3′ end, becomes covalently linked to the target plasmid in the strand transfer reaction.


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

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

The 3′ end of ISY100 becomes covalently linked to the target DNA in the strand transfer reaction. A. A pre-cleaved transposon end, 5′ end-labelled on either the top or bottom strand, was incubated with transposase and a supercoiled target plasmid. Samples were withdrawn at the indicated times and run on a non-denaturing agarose gel (TAE) or a strand-separating alkaline gel (NaOH). DNA was detected by ethidium staining, and by autoradiography (32P). The positions of supercoiled (sc) and open circle (oc) target plasmid are shown to the left of the ethidium-stained gel. The positions of single end-joined (SEJ) and double end-joined (DEJ) strand transfer products are shown to the right of the autoradiographs. SEJ and DEJ products are indistinguishable on the strand separating gel. Unreacted labelled substrate has been cropped from the bottom of both autoradiographs. B. Sequence of the pre-cleaved end and schematic diagram of the reaction products.
© Copyright Policy
Related In: Results  -  Collection

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

fig08: The 3′ end of ISY100 becomes covalently linked to the target DNA in the strand transfer reaction. A. A pre-cleaved transposon end, 5′ end-labelled on either the top or bottom strand, was incubated with transposase and a supercoiled target plasmid. Samples were withdrawn at the indicated times and run on a non-denaturing agarose gel (TAE) or a strand-separating alkaline gel (NaOH). DNA was detected by ethidium staining, and by autoradiography (32P). The positions of supercoiled (sc) and open circle (oc) target plasmid are shown to the left of the ethidium-stained gel. The positions of single end-joined (SEJ) and double end-joined (DEJ) strand transfer products are shown to the right of the autoradiographs. SEJ and DEJ products are indistinguishable on the strand separating gel. Unreacted labelled substrate has been cropped from the bottom of both autoradiographs. B. Sequence of the pre-cleaved end and schematic diagram of the reaction products.
Mentions: To investigate the strand transfer activity of transposase, we used double-stranded oligonucleotides representing cleaved transposon ends. The termini were chosen to be the same as those produced on supercoiled plasmid substrates, exactly at the transposon 3′ end and two nucleotides inside the transposon at the 5′ end. Pre-cleaved transposon ends were labelled on either the top or bottom strand, and incubated together with a supercoiled target plasmid and transposase in a time course experiment. The products were run on a non-denaturing agarose gel and detected by autoradiography. Radioactive products co-migrating with the open circular and linear forms of the target plasmid were produced only in the presence of transposase, divalent metal ions and target plasmid (Fig. 8A and data not shown). The mobilities of these products are consistent with transfer of one or two transposon ends to a target plasmid, yielding nicked circle with one attached transposon end, and a linear molecule with two attached transposon ends respectively (Fig. 8B). Strand transfer products accumulated over time, and were observed irrespective of whether the top or the bottom strand was labelled (Fig. 8A). When the same reactions were run on a denaturing agarose gel, plasmid-sized labelled products were observed only when the transposon end was labelled on the bottom strand (Fig. 8A). Thus only the bottom strand, containing the transposon 3′ end, becomes covalently linked to the target plasmid in the strand transfer reaction.

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