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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

Multiple pathways for the unfolding and cleavage of the Tn10 transpososome. The model summarizes the potential of the transpososome to unfold at different stages of the cleavage reaction. At the start of the reaction, the bPEC can follow one of the three pathways. The least productive is via the βSEB intermediate. Transpososomes that arrive at the DEB stage of the reaction with IHF still associated, or reacquire IHF at this stage of the reaction, unfold and perform the strand transfer step very slowly.
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fig7: Multiple pathways for the unfolding and cleavage of the Tn10 transpososome. The model summarizes the potential of the transpososome to unfold at different stages of the cleavage reaction. At the start of the reaction, the bPEC can follow one of the three pathways. The least productive is via the βSEB intermediate. Transpososomes that arrive at the DEB stage of the reaction with IHF still associated, or reacquire IHF at this stage of the reaction, unfold and perform the strand transfer step very slowly.

Mentions: The IHF-loop has an important structural role during assembly of the Tn10 transpososome. However, it is also required to promote conformational changes during the cleavage steps of the reaction. This was demonstrated previously because disruption of the loop blocks resolution of the DNA hairpin intermediate on the first transposon end to be cleaved and stalls the reaction at the SEB stage (26). The results presented here provide further insight into this mechanism by showing that there are multiple pathways for unfolding of the IHF-loop and cleavage of the transposon ends. The pathways are summarized in Figure 7 where they are illustrated for a mixed complex that represents transposition of IS10 in which only one transposon end has an IHF binding site.


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)

Multiple pathways for the unfolding and cleavage of the Tn10 transpososome. The model summarizes the potential of the transpososome to unfold at different stages of the cleavage reaction. At the start of the reaction, the bPEC can follow one of the three pathways. The least productive is via the βSEB intermediate. Transpososomes that arrive at the DEB stage of the reaction with IHF still associated, or reacquire IHF at this stage of the reaction, unfold and perform the strand transfer step very slowly.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1074725&req=5

fig7: Multiple pathways for the unfolding and cleavage of the Tn10 transpososome. The model summarizes the potential of the transpososome to unfold at different stages of the cleavage reaction. At the start of the reaction, the bPEC can follow one of the three pathways. The least productive is via the βSEB intermediate. Transpososomes that arrive at the DEB stage of the reaction with IHF still associated, or reacquire IHF at this stage of the reaction, unfold and perform the strand transfer step very slowly.
Mentions: The IHF-loop has an important structural role during assembly of the Tn10 transpososome. However, it is also required to promote conformational changes during the cleavage steps of the reaction. This was demonstrated previously because disruption of the loop blocks resolution of the DNA hairpin intermediate on the first transposon end to be cleaved and stalls the reaction at the SEB stage (26). The results presented here provide further insight into this mechanism by showing that there are multiple pathways for unfolding of the IHF-loop and cleavage of the transposon ends. The pathways are summarized in Figure 7 where they are illustrated for a mixed complex that represents transposition of IS10 in which only one transposon end has an IHF binding site.

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