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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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Related in: MedlinePlus

Kinetics and requirements for unfolding of the SEB isomers. Mixed complexes were assembled using the transposon ends illustrated below each panel. There was a 4-fold molar excess of the outside end present. The outside end was prepared by digesting pRC98 with AccI+ScaI (84 bp transposon arm/75 bp flanking DNA). The precleaved outside end was prepared by digesting pRC35 with BstEII+PvuII (85 bp transposon arm). The even-end DNA fragment was prepared by digesting pRC100 with AccI+BamHI (73 bp transposon arm/39 bp flanking DNA). The precleaved even-end was prepared by digesting pRC99 with AccI+PvuII (73 bp transposon arm). Other details are as given in Figure 2.
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fig4: Kinetics and requirements for unfolding of the SEB isomers. Mixed complexes were assembled using the transposon ends illustrated below each panel. There was a 4-fold molar excess of the outside end present. The outside end was prepared by digesting pRC98 with AccI+ScaI (84 bp transposon arm/75 bp flanking DNA). The precleaved outside end was prepared by digesting pRC35 with BstEII+PvuII (85 bp transposon arm). The even-end DNA fragment was prepared by digesting pRC100 with AccI+BamHI (73 bp transposon arm/39 bp flanking DNA). The precleaved even-end was prepared by digesting pRC99 with AccI+PvuII (73 bp transposon arm). Other details are as given in Figure 2.

Mentions: To further confirm the identity of the SEB complexes and to validate their surprising unfolding behavior, the two isomers of the SEB complex were prepared directly by mixing outside end and even-end DNA fragments (Figure 4). Recall that the absence of an IHF binding site on the even-end ensures that it is always incorporated on the β side of the complex. As before, the radioactive label was present only on the even-end, so that only mixed complexes are detected. The sfβSEB was prepared by mixing an uncleaved outside end with a precleaved even-end (left panel). Counter wise, the sfαSEB was prepared by mixing an uncleaved even-end and a precleaved outside end (right panel). When the complexes are challenged with Ca++ to unlock the α side of the complex and with heparin to strip out the IHF, only the αSEB complex unfolds. The βSEB complex was highly resistant to unfolding and 80% remained after overnight incubation (Figure 4, left panel). These results confirm the unfolding behavior shown in Figure 3 where the αSEB and βSEB complexes were prepared in a single mixture.


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)

Kinetics and requirements for unfolding of the SEB isomers. Mixed complexes were assembled using the transposon ends illustrated below each panel. There was a 4-fold molar excess of the outside end present. The outside end was prepared by digesting pRC98 with AccI+ScaI (84 bp transposon arm/75 bp flanking DNA). The precleaved outside end was prepared by digesting pRC35 with BstEII+PvuII (85 bp transposon arm). The even-end DNA fragment was prepared by digesting pRC100 with AccI+BamHI (73 bp transposon arm/39 bp flanking DNA). The precleaved even-end was prepared by digesting pRC99 with AccI+PvuII (73 bp transposon arm). Other details are as given in Figure 2.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1074725&req=5

fig4: Kinetics and requirements for unfolding of the SEB isomers. Mixed complexes were assembled using the transposon ends illustrated below each panel. There was a 4-fold molar excess of the outside end present. The outside end was prepared by digesting pRC98 with AccI+ScaI (84 bp transposon arm/75 bp flanking DNA). The precleaved outside end was prepared by digesting pRC35 with BstEII+PvuII (85 bp transposon arm). The even-end DNA fragment was prepared by digesting pRC100 with AccI+BamHI (73 bp transposon arm/39 bp flanking DNA). The precleaved even-end was prepared by digesting pRC99 with AccI+PvuII (73 bp transposon arm). Other details are as given in Figure 2.
Mentions: To further confirm the identity of the SEB complexes and to validate their surprising unfolding behavior, the two isomers of the SEB complex were prepared directly by mixing outside end and even-end DNA fragments (Figure 4). Recall that the absence of an IHF binding site on the even-end ensures that it is always incorporated on the β side of the complex. As before, the radioactive label was present only on the even-end, so that only mixed complexes are detected. The sfβSEB was prepared by mixing an uncleaved outside end with a precleaved even-end (left panel). Counter wise, the sfαSEB was prepared by mixing an uncleaved even-end and a precleaved outside end (right panel). When the complexes are challenged with Ca++ to unlock the α side of the complex and with heparin to strip out the IHF, only the αSEB complex unfolds. The βSEB complex was highly resistant to unfolding and 80% remained after overnight incubation (Figure 4, left panel). These results confirm the unfolding behavior shown in Figure 3 where the αSEB and βSEB complexes were prepared in a single mixture.

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