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Cyclic changes in the affinity of protein-DNA interactions drive the progression and regulate the outcome of the Tn10 transposition reaction.

Liu D, Crellin P, Chalmers R - Nucleic Acids Res. (2005)

Bottom Line: During transpososome assembly, IHF is bound with high affinity.However, the affinity for IHF drops dramatically after cleavage of the first transposon end, leading to IHF ejection and unfolding of the complex.The ejection of IHF promotes cleavage of the second end, which is followed by restoration of the high affinity state which in turn regulates target interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford South Parks Road, Oxford OX1 3QU, UK.

ABSTRACT
The Tn10 transpososome is a DNA processing machine in which two transposon ends, a transposase dimer and the host protein integration host factor (IHF), are united in an asymmetrical complex. The transitions that occur during one transposition cycle are not limited to chemical cleavage events at the transposon ends, but also involve a reorganization of the protein and DNA components. Here, we demonstrate multiple pathways for Tn10 transposition. We show that one series of events is favored over all others and involves cyclic changes in the affinity of IHF for its binding site. During transpososome assembly, IHF is bound with high affinity. However, the affinity for IHF drops dramatically after cleavage of the first transposon end, leading to IHF ejection and unfolding of the complex. The ejection of IHF promotes cleavage of the second end, which is followed by restoration of the high affinity state which in turn regulates target interactions.

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Insertion kinetics of the different complexes. (A–D) and (F–K) The standard 20 μl reaction was scaled up to 200 μl and initiated at time zero by the addition of Mg++ in a mixture with 10 μg of pBluescript as target DNA. Aliquots (40 μl) were withdrawn at the indicated time and stopped by the addition of EDTA. Half of the reaction was deproteinated by SDS treatment to allow detection of the large insertion product. For each type of complex, the native and deproteinated aliquots (20 μl) were electrophoresed on the same gel so that the quantification of the various complexes and products was exactly comparable and did not require normalization. The sequence of bases flanking the transposon ends on each fragment was isogenic to preclude any potential effect on the rate of cleavage. bPEC panel; the outside end was prepared by digesting pRC98 with AccI+SacII (84 bp transposon arm/75 bp flanking DNA). αSEB panel: the precleaved outside end was prepared by digesting pRC35 with BstEII+PvuII (85 bp transposon arm); the uncleaved even-end DNA fragment was prepared by digesting pRC100 with AccI+BamHI (73 bp transposon arm/39 bp flanking DNA). βSEB panel: the uncleaved outside end was prepared by digesting pRC98 with AccI+SacII (84 bp transposon arm/75 bp flanking DNA); the precleaved even-end was prepared by digesting pRC99 with AccI+PvuII (73 bp transposon arm). DEB panel: the precleaved outside end was prepared by digesting pRC35 with BstEII+PvuII (85 bp transposon arm). (E) Transposition reactions (200 μl) contained 1 nM plasmid substrate encoding a mini-Tn10 transposon, 6 nM transposase and 35 nM IHF. The reactions were incubated at 30°C for 3 h, concentrated by ethanol precipitation and analyzed on a TBE-buffered 1.1% agarose gel (30,39). Excision of the transposon segment from the supercoiled plasmid substrate produces the DEB product (referred to in previous publications as the ‘excised transposon fragment’ or ETF). Auto-integration of the DEB product produces a topologically complex set of knot and catenane products (39). Apart from the DEB product, these are the only products present in the section of the gel shown. The IHF-up and IHF-down mutations were encoded on plasmids pNK2588 and pNK2590, respectively. IHF-up (single point mutation) and IHF-down (triple point mutation) move the IHF binding site closer to or further away from the IHF consensus binding site as described in Figure 1 of ref. (46). A reverse contrast photograph of an ethidium bromide stained agarose gel is shown.
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fig6: Insertion kinetics of the different complexes. (A–D) and (F–K) The standard 20 μl reaction was scaled up to 200 μl and initiated at time zero by the addition of Mg++ in a mixture with 10 μg of pBluescript as target DNA. Aliquots (40 μl) were withdrawn at the indicated time and stopped by the addition of EDTA. Half of the reaction was deproteinated by SDS treatment to allow detection of the large insertion product. For each type of complex, the native and deproteinated aliquots (20 μl) were electrophoresed on the same gel so that the quantification of the various complexes and products was exactly comparable and did not require normalization. The sequence of bases flanking the transposon ends on each fragment was isogenic to preclude any potential effect on the rate of cleavage. bPEC panel; the outside end was prepared by digesting pRC98 with AccI+SacII (84 bp transposon arm/75 bp flanking DNA). αSEB panel: the precleaved outside end was prepared by digesting pRC35 with BstEII+PvuII (85 bp transposon arm); the uncleaved even-end DNA fragment was prepared by digesting pRC100 with AccI+BamHI (73 bp transposon arm/39 bp flanking DNA). βSEB panel: the uncleaved outside end was prepared by digesting pRC98 with AccI+SacII (84 bp transposon arm/75 bp flanking DNA); the precleaved even-end was prepared by digesting pRC99 with AccI+PvuII (73 bp transposon arm). DEB panel: the precleaved outside end was prepared by digesting pRC35 with BstEII+PvuII (85 bp transposon arm). (E) Transposition reactions (200 μl) contained 1 nM plasmid substrate encoding a mini-Tn10 transposon, 6 nM transposase and 35 nM IHF. The reactions were incubated at 30°C for 3 h, concentrated by ethanol precipitation and analyzed on a TBE-buffered 1.1% agarose gel (30,39). Excision of the transposon segment from the supercoiled plasmid substrate produces the DEB product (referred to in previous publications as the ‘excised transposon fragment’ or ETF). Auto-integration of the DEB product produces a topologically complex set of knot and catenane products (39). Apart from the DEB product, these are the only products present in the section of the gel shown. The IHF-up and IHF-down mutations were encoded on plasmids pNK2588 and pNK2590, respectively. IHF-up (single point mutation) and IHF-down (triple point mutation) move the IHF binding site closer to or further away from the IHF consensus binding site as described in Figure 1 of ref. (46). A reverse contrast photograph of an ethidium bromide stained agarose gel is shown.

Mentions: All of the unfolding experiments described above were performed under non-catalytic conditions. We therefore sought to determine whether the unfolding kinetics of the various complexes is correlated with the efficiency of the insertion reaction (Figure 6). A single large reaction was assembled for each of the complexes and initiated at time zero by addition of the catalytic metal ion in a mixture with a closed circular target plasmid. The target plasmid also served as a competitor that will sequester IHF after a single cycle of dissociation from the transpososome. At each time point, an aliquot was removed from the reaction and stopped by the addition of EDTA to chelate the Mg++. Half of each aliquot was used to visualize the complexes, while the other half was deproteinated by SDS treatment to allow detection of the large insertion product. For each type of complex, the native and denatured sets of samples were electrophoresed on the same gel so that the quantification of the native complexes and deproteinated insertion products would be directly comparable.


Cyclic changes in the affinity of protein-DNA interactions drive the progression and regulate the outcome of the Tn10 transposition reaction.

Liu D, Crellin P, Chalmers R - Nucleic Acids Res. (2005)

Insertion kinetics of the different complexes. (A–D) and (F–K) The standard 20 μl reaction was scaled up to 200 μl and initiated at time zero by the addition of Mg++ in a mixture with 10 μg of pBluescript as target DNA. Aliquots (40 μl) were withdrawn at the indicated time and stopped by the addition of EDTA. Half of the reaction was deproteinated by SDS treatment to allow detection of the large insertion product. For each type of complex, the native and deproteinated aliquots (20 μl) were electrophoresed on the same gel so that the quantification of the various complexes and products was exactly comparable and did not require normalization. The sequence of bases flanking the transposon ends on each fragment was isogenic to preclude any potential effect on the rate of cleavage. bPEC panel; the outside end was prepared by digesting pRC98 with AccI+SacII (84 bp transposon arm/75 bp flanking DNA). αSEB panel: the precleaved outside end was prepared by digesting pRC35 with BstEII+PvuII (85 bp transposon arm); the uncleaved even-end DNA fragment was prepared by digesting pRC100 with AccI+BamHI (73 bp transposon arm/39 bp flanking DNA). βSEB panel: the uncleaved outside end was prepared by digesting pRC98 with AccI+SacII (84 bp transposon arm/75 bp flanking DNA); the precleaved even-end was prepared by digesting pRC99 with AccI+PvuII (73 bp transposon arm). DEB panel: the precleaved outside end was prepared by digesting pRC35 with BstEII+PvuII (85 bp transposon arm). (E) Transposition reactions (200 μl) contained 1 nM plasmid substrate encoding a mini-Tn10 transposon, 6 nM transposase and 35 nM IHF. The reactions were incubated at 30°C for 3 h, concentrated by ethanol precipitation and analyzed on a TBE-buffered 1.1% agarose gel (30,39). Excision of the transposon segment from the supercoiled plasmid substrate produces the DEB product (referred to in previous publications as the ‘excised transposon fragment’ or ETF). Auto-integration of the DEB product produces a topologically complex set of knot and catenane products (39). Apart from the DEB product, these are the only products present in the section of the gel shown. The IHF-up and IHF-down mutations were encoded on plasmids pNK2588 and pNK2590, respectively. IHF-up (single point mutation) and IHF-down (triple point mutation) move the IHF binding site closer to or further away from the IHF consensus binding site as described in Figure 1 of ref. (46). A reverse contrast photograph of an ethidium bromide stained agarose gel is shown.
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Related In: Results  -  Collection

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fig6: Insertion kinetics of the different complexes. (A–D) and (F–K) The standard 20 μl reaction was scaled up to 200 μl and initiated at time zero by the addition of Mg++ in a mixture with 10 μg of pBluescript as target DNA. Aliquots (40 μl) were withdrawn at the indicated time and stopped by the addition of EDTA. Half of the reaction was deproteinated by SDS treatment to allow detection of the large insertion product. For each type of complex, the native and deproteinated aliquots (20 μl) were electrophoresed on the same gel so that the quantification of the various complexes and products was exactly comparable and did not require normalization. The sequence of bases flanking the transposon ends on each fragment was isogenic to preclude any potential effect on the rate of cleavage. bPEC panel; the outside end was prepared by digesting pRC98 with AccI+SacII (84 bp transposon arm/75 bp flanking DNA). αSEB panel: the precleaved outside end was prepared by digesting pRC35 with BstEII+PvuII (85 bp transposon arm); the uncleaved even-end DNA fragment was prepared by digesting pRC100 with AccI+BamHI (73 bp transposon arm/39 bp flanking DNA). βSEB panel: the uncleaved outside end was prepared by digesting pRC98 with AccI+SacII (84 bp transposon arm/75 bp flanking DNA); the precleaved even-end was prepared by digesting pRC99 with AccI+PvuII (73 bp transposon arm). DEB panel: the precleaved outside end was prepared by digesting pRC35 with BstEII+PvuII (85 bp transposon arm). (E) Transposition reactions (200 μl) contained 1 nM plasmid substrate encoding a mini-Tn10 transposon, 6 nM transposase and 35 nM IHF. The reactions were incubated at 30°C for 3 h, concentrated by ethanol precipitation and analyzed on a TBE-buffered 1.1% agarose gel (30,39). Excision of the transposon segment from the supercoiled plasmid substrate produces the DEB product (referred to in previous publications as the ‘excised transposon fragment’ or ETF). Auto-integration of the DEB product produces a topologically complex set of knot and catenane products (39). Apart from the DEB product, these are the only products present in the section of the gel shown. The IHF-up and IHF-down mutations were encoded on plasmids pNK2588 and pNK2590, respectively. IHF-up (single point mutation) and IHF-down (triple point mutation) move the IHF binding site closer to or further away from the IHF consensus binding site as described in Figure 1 of ref. (46). A reverse contrast photograph of an ethidium bromide stained agarose gel is shown.
Mentions: All of the unfolding experiments described above were performed under non-catalytic conditions. We therefore sought to determine whether the unfolding kinetics of the various complexes is correlated with the efficiency of the insertion reaction (Figure 6). A single large reaction was assembled for each of the complexes and initiated at time zero by addition of the catalytic metal ion in a mixture with a closed circular target plasmid. The target plasmid also served as a competitor that will sequester IHF after a single cycle of dissociation from the transpososome. At each time point, an aliquot was removed from the reaction and stopped by the addition of EDTA to chelate the Mg++. Half of each aliquot was used to visualize the complexes, while the other half was deproteinated by SDS treatment to allow detection of the large insertion product. For each type of complex, the native and denatured sets of samples were electrophoresed on the same gel so that the quantification of the native complexes and deproteinated insertion products would be directly comparable.

Bottom Line: During transpososome assembly, IHF is bound with high affinity.However, the affinity for IHF drops dramatically after cleavage of the first transposon end, leading to IHF ejection and unfolding of the complex.The ejection of IHF promotes cleavage of the second end, which is followed by restoration of the high affinity state which in turn regulates target interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford South Parks Road, Oxford OX1 3QU, UK.

ABSTRACT
The Tn10 transpososome is a DNA processing machine in which two transposon ends, a transposase dimer and the host protein integration host factor (IHF), are united in an asymmetrical complex. The transitions that occur during one transposition cycle are not limited to chemical cleavage events at the transposon ends, but also involve a reorganization of the protein and DNA components. Here, we demonstrate multiple pathways for Tn10 transposition. We show that one series of events is favored over all others and involves cyclic changes in the affinity of IHF for its binding site. During transpososome assembly, IHF is bound with high affinity. However, the affinity for IHF drops dramatically after cleavage of the first transposon end, leading to IHF ejection and unfolding of the complex. The ejection of IHF promotes cleavage of the second end, which is followed by restoration of the high affinity state which in turn regulates target interactions.

Show MeSH
Related in: MedlinePlus