The positive and negative regulation of Tn10 transposition by IHF is mediated by structurally asymmetric transposon arms

Sewitz, Sven; Crellin, Paul K.; Chalmers, Ronald

doi:10.1093/nar/gkg797

Cited by 25 publications

(39 citation statements)

References 44 publications

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“…Second, IHF is unusual among DNA-binding proteins for its lack of salt dependence over the range of concentrations used in all of the experiments reported (26). 5 Third, where hard data exist, reported values were corrected to a standard set of reaction conditions. ⌬G 0 values calculated from K d values reported were corrected to a standard temperature (20°C) using the standard thermodynamic relationship as shown in Equation 9, where K a,ref and K a,T refer to the association equilibrium constants at the reference (293 K) and experimental temperatures, (Eq.…”

Section: Analysis Of Naturally Occurring Ihf-binding Sites-mentioning

confidence: 99%

“…Bending plays an architectural role in the primary role for IHF in chromosome condensation (3). However, unlike other class members that exhibit no sequence specificity in DNA binding, IHF also binds in a sequence-specific manner to sites at which bending aids in the formation of higher order structures required for a variety of cellular functions such as site-specific recombination (4), transposition of mobile genetic elements (5), gene regulation (6), and DNA replication (7).…”

mentioning

confidence: 99%

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Indirect Recognition in Sequence-specific DNA Binding by Escherichia coli Integration Host Factor

Aeling

Opel

Steffen

et al. 2006

Journal of Biological Chemistry

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Integration host factor (IHF) is a bacterial histone-like protein whose primary biological role is to condense the bacterial nucleoid and to constrain DNA supercoils. It does so by binding in a sequence-independent manner throughout the genome. However, unlike other structurally related bacterial histone-like proteins, IHF has evolved a sequence-dependent, high affinity DNA-binding motif. The high affinity binding sites are important for the regulation of a wide range of cellular processes. A remarkable feature of IHF is that it employs an indirect readout mechanism to bind and wrap DNA at both the nonspecific and high affinity (sequence-dependent) DNA sites. In this study we assessed the contributions of pre-formed and protein-induced DNA conformations to the energetics of IHF binding. Binding energies determined experimentally were compared with energies predicted for the IHF-induced deformation of the DNA helix (DNA deformation energy) in the IHF-DNA complex. Combinatorial sets of de novo DNA sequences were designed to systematically evaluate the influence of sequence-dependent structural characteristics of the conserved IHF recognition elements of the consensus DNA sequence. We show that IHF recognizes pre-formed conformational characteristics of the consensus DNA sequence at high affinity sites, whereas at all other sites relative affinity is determined by the deformational energy required for nearest-neighbor base pairs to adopt the DNA structure of the bound DNA-IHF complex.Site-specific DNA binding by regulatory proteins is a feature of the regulatory processes that maintain, expand, and express genetic information such as replication, recombination, transposition, and transcription. The chemical and physical mechanisms that underlie sequence-specific recognition of regulatory elements by cognate DNA-binding proteins are typically classified as direct versus indirect readout. The former refers primarily to hydrogen bonds between proteins and the unique extra-cyclic substituents at C-4 of pyrimidines, C-6 of purines, and N-7 of purines. These groups provide a base pair-specific pattern of hydrogen bond donors and acceptors in the major groove of DNA that can be directly read by a complementary pattern of amino acid side chain donors and acceptors. Indirect readout refers to recognition of aspects of DNA structure such as intrinsic curvature, topology of major and minor grooves, ordered water structures, local geometry of backbone phosphates, and flexibility or deformability. Because both the local DNA structure and energy to deform DNA are themselves intrinsic sequence-dependent properties, the conserved sequences that distinguish binding sites necessarily include contributions from both direct and indirect mechanisms. Consequently, although the contribution from indirect mechanisms is expected to be significant in protein-DNA complexes that feature substantial DNA deformation, it has proven difficult to evaluate these contributions quantitatively. A protein that relies exclusively, or primarily, on indir...

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Section: Analysis Of Naturally Occurring Ihf-binding Sites-mentioning

confidence: 99%

mentioning

confidence: 99%

Indirect Recognition in Sequence-specific DNA Binding by Escherichia coli Integration Host Factor

Aeling

Opel

Steffen

et al. 2006

Journal of Biological Chemistry

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“…Enhanced OP-Cu cleavage is also apparent at nucleotides 2-5 of the transferred strand and nucleotides 3-6 of the nontransferred strand. There is no clear region of protection over the IHF site of the folded transpososome, probably because IHF is bound tightly to only one OE (Sewitz et al 2003). …”

Section: H-ns Binds To Donor Dna and Induces Transpososome Unfoldingmentioning

confidence: 99%

The global regulator H-NS acts directly on the transpososome to promote Tn10 transposition

Wardle¹,

O'Carroll²,

Derbyshire³

et al. 2005

Genes Dev.

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The histone-like nucleoid structuring (H-NS) protein is a global transcriptional regulator that is known to regulate stress response pathways and virulence genes in bacteria. It has also been implicated in the regulation of bacterial transposition systems, including Tn10. We demonstrate here that H-NS promotes Tn10 transposition by binding directly to the transposition complex (or transpososome). We present evidence that, upon binding, H-NS induces the unfolding of the Tn10 transpososome and helps to maintain the transpososome in an unfolded state. This ensures that intermolecular (as opposed to self-destructive intramolecular) transposition events are favored. We present evidence that H-NS binding to the flanking donor DNA of the transpososome is the initiating event in the unfolding process. We propose that by recruiting H-NS as a modulator of transposition, Tn10 has evolved a means of sensing changes in host physiology, as the amount of H-NS in the cell, as well its activity, are responsive to changes in environmental conditions. Sensing of environmental changes through H-NS would allow transposition to occur when it is most opportune for both the transposon and the host.

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“…The reactions (20 l) contained 80 fmol of transposase (4 nM) and 37 fmol of transposon end. The reaction mixture was treated with hydroxyl radicals to generate a footprint as described previously (15,51). Complex 1, complex 2, and the free DNA from nine such reactions were purified in an EMSA and pooled, and the footprint was displayed on a DNA sequencing gel.…”

Section: Vol 24 2004mentioning

confidence: 99%

Early Intermediates of mariner Transposition: Catalysis without Synapsis of the Transposon Ends Suggests a Novel Architecture of the Synaptic Complex

Lipkow

Buisine

Lampe

et al. 2004

Molecular and Cellular Biology

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The mariner family is probably the most widely distributed family of transposons in nature. Although these transposons are related to the well-studied bacterial insertion elements, there is evidence for major differences in their reaction mechanisms. We report the identification and characterization of complexes that contain the Himar1 transposase bound to a single transposon end. Titrations and mixing experiments with the native transposase and transposase fusions suggested that they contain different numbers of transposase monomers. However, the DNA protection footprints of the two most abundant single-end complexes are identical. This indicates that some transposase monomers may be bound to the transposon end solely by protein-protein interactions. This would mean that the Himar1 transposase can dimerize independently of the second transposon end and that the architecture of the synaptic complex has more in common with V(D)J recombination than with bacterial insertion elements. Like V(D)J recombination and in contrast to the case for bacterial elements, Himar1 catalysis does not appear to depend on synapsis of the transposon ends, and the single-end complexes are active for nicking and probably for cleavage. We discuss the role of this single-end activity in generating the mutations that inactivate the vast majority of mariner elements in eukaryotes.The Tc1/mariner superfamily of transposons consists of the Tc1 and mariner families of elements in eukaryotes and the more distantly related IS630-like elements in bacteria (25, 39). Members of the superfamily have a single transposase gene expressed in the germ line and/or the soma, transpose via a "cut and paste" DNA intermediate, and duplicate a TA dinucleotide upon insertion. This is probably the most widespread family of transposons in nature: members have been identified in bacteria (IS630), ciliates, fungi, plants, and most animal phyla, from Porifera (sponges) to humans. Although mariner elements are widespread, they are unevenly distributed in closely related species and the vast majority are inactive because of mutations. This suggests that they have an unusual life style that involves a high rate of horizontal transfer to new hosts, followed by a burst of transposition and subsequent vertical inactivation (37, 43).Homology-dependent gene silencing serves to control the spread of transposons, retroviruses, and other repetitive DNA elements in many eukaryotes. It is mediated by at least three distinct mechanisms, specifically DNA methylation, histone deacetylation, and RNA interference. However, depending on the identity of the organism and the repetitive element in question, these mechanisms are not always completely effective. As noted by McClintock, the rate of transposition in a given germ line can change over time, with cycles of activation and silencing lasting several generations (cited in reference 35). Furthermore, although all three mechanisms of homologydependent gene silencing operate in plants and are active against many retrotransposons, som...

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The positive and negative regulation of Tn10 transposition by IHF is mediated by structurally asymmetric transposon arms

Cited by 25 publications

References 44 publications

Indirect Recognition in Sequence-specific DNA Binding by Escherichia coli Integration Host Factor

Indirect Recognition in Sequence-specific DNA Binding by Escherichia coli Integration Host Factor

The global regulator H-NS acts directly on the transpososome to promote Tn10 transposition

Early Intermediates of mariner Transposition: Catalysis without Synapsis of the Transposon Ends Suggests a Novel Architecture of the Synaptic Complex

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