ORCID Profile
0000-0002-1395-7173
Current Organisation
University of Nottingham
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Publisher: Elsevier BV
Date: 02-2004
DOI: 10.1016/S1097-2765(04)00052-8
Abstract: DNA loops and bends are common features of DNA processing machines. The bacterial transposon Tn10 has recruited integration host factor (IHF), a site-specific DNA-bending protein, as an architectural component for assembly of the higher-order nucleoprotein complex within which the transposition reaction takes place. Here, we demonstrate additional roles for the IHF loop during the catalytic steps of the reaction. We show that metal ion-dependent unfolding of the IHF-bent transposon arm is communicated to the catalytic center, inducing a substantial conformational change in the DNA. Partial disruption of the IHF loop shows that this step promotes resolution of the hairpin intermediate on one transposon end and initiation of catalysis at the other. Further evidence suggests that the molecular mechanism responsible may be mechanical stress in the IHF loop, related to a change in the relative position of the transposase contacts that anchor the loop on either side.
Publisher: American Society for Microbiology
Date: 15-05-2000
DOI: 10.1128/JB.182.10.2970-2972.2000
Abstract: The complete nucleotide sequence of Tn 10 has been determined. The dinucleotide signature and percent G+C of the sequence had no discontinuities, indicating that Tn 10 constitutes a homogeneous unit. The new sequence contained three new open reading frames corresponding to a glutamate permease, repressors of heavy metal resistance operons, and a hypothetical protein in Bacillus subtilis . The glutamate permease was fully functional when expressed, but Tn 10 did not protect Escherichia coli from the toxic effects of various metals.
Publisher: Oxford University Press (OUP)
Date: 15-10-2003
DOI: 10.1093/NAR/GKG797
Abstract: The Tn10 transpososome has symmetrical components on either side: there are two transposon ends each of which has binding sites for a monomer of transposase and an IHF heterodimer. The DNA bending activity of IHF stimulates assembly of an intermediate with tightly folded transposon ends in which transposase has additional 'subterminal' DNA contacts, located distal to the IHF site. These subterminal contacts are required to activate later steps in the reaction. Quantitative hydroxyl radical footprinting and gel retardation unfolding experiments show that the transpososome is fundamentally asymmetric, despite having identical components on either side. Major differences between the transposon ends define alpha and beta sides of the complex. IHF can dissociate from the transposon arm on the beta side of the complex in the absence of metal ion. However, IHF is locked onto the alpha side of the complex, probably by the subterminal transposase contacts, until released by a metal ion-dependent conformational change. Later in the reaction, IHF inhibits target interactions. Using a very short transposon arm, target interactions are demonstrated at a saturating IHF concentration. This suggests that inhibition of target interactions is due to steric hindrance of the target binding site by a single IHF-folded transposon arm.
Publisher: Oxford University Press (OUP)
Date: 23-03-2005
DOI: 10.1093/NAR/GKI348
Publisher: Wiley
Date: 16-07-2001
Publisher: Wiley
Date: 02-11-2006
DOI: 10.1111/J.1365-2958.2006.05432.X
Abstract: DNA processing reactions often involve multiple components acting in concert to achieve the desired outcome. However, it is usually difficult to know how the components communicate and cooperate to orchestrate an ordered series of events. We address this question in the context of the Tn 10 transposition reaction, in which the DNA cleavage and joining events occur within a higher-order complex containing a transposase dimer, two transposon ends and the DNA-bending host-factor IHF (Integration Host Factor). Previously it was shown that the complex is asymmetric. The a side consists of an IHF protomer initially immobilized by a DNA-loop, but subsequently used to promote conformational changes required for the cleavage steps. The beta side of the complex was considered to fulfil a more passive role. Here we show that the a side of the complex promotes coupled conformational changes at both transposon ends, while the a and beta sides communicate and cooperate to dominate different phases of the transposition reaction. Together, these effects provide for a robust response to critical changes in the transposon end. These findings also explain the intriguing genetic phenotypes of a series of previously reported Tn10 mutants and have consequences for the evolution of new elements.
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for ronald chalmers.