DNA-binding Parameters of the HU Protein of Escherichia coli to Cruciform DNA

Bonnefoy, Eliette; Takahashi, Masayuki; Yaniv, Josette Rouvière

doi:10.1006/jmbi.1994.1563

Cited by 135 publications

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“…For example, the integration host factor interacts with its specific sites with high affinity (K d Ϸ 50 nM) and with generic DNA with a K d of about 20 M (39). Instead, H-NS interacts with DNA with similar affinity to HU, the other major chromatin-associated protein in E. coli, which has a K d for generic DNA molecules of about 1 M (40). The low affinity of the H-NS-DNA interaction cannot be reconciled with a classical repressor-type mechanism for modulating gene expression.…”

Section: Discussionmentioning

confidence: 88%

DNA Binding Is Not Sufficient for H-NS-mediated Repression ofproU Expression

Jordi¹,

Fielder

Burns

et al. 1997

Journal of Biological Chemistry

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H-NS is a major component of bacterial chromatin and influences the expression of many genes. H-NS has beenshown to exhibit a binding preference for certain ATrich curved DNA elements in vitro. In this study we have addressed the factors that determine the specificity of H-NS action in vitro and in vivo. In bandshift studies, H-NS showed a slight binding preference for all curved sequences tested whether GC-based or AT-based; the specific architecture of the curve also influenced H-NS binding. In filter retention assays little difference in affinity could be detected for any sequence tested, including the downstream regulatory element (DRE) a downstream curved DNA element required for H-NS to repress transcription of the Salmonella typhimurium proU operon in vivo. A K d of 1-2 M was estimated for binding of H-NS to each of these sequences. In vivo, the distance between the proU promoter and the DRE, their relative orientations on the face of the DNA helix, and translation of the DRE had no major effect on proU regulation. None of the synthetic curved sequences tested could functionally replace the DRE in vivo. These data show that differential binding to curved DNA cannot account for the specificity of H-NS action in vivo. Furthermore, binding of H-NS to DNA per se is insufficient to repress the proU promoter. Thus, the DRE does not simply act as an H-NS binding site but must have a more specific role in mediating H-NS regulation of proU transcription.

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Section: Discussionmentioning

confidence: 88%

DNA Binding Is Not Sufficient for H-NS-mediated Repression ofproU Expression

Jordi¹,

Fielder

Burns

et al. 1997

Journal of Biological Chemistry

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“…Fig. 1D, DNA 1; [37]] was incubated with HMGB1(A+B) and complexes were examined by gel electrophoresis (Fig. 2B).…”

Section: Resultsmentioning

confidence: 99%

“…Some oligonucleotides were labeled with[γ-32 P]ATP and T4 polynucleotide kinase (New England Biolabs). Cruciform DNA was prepared as described [37]. Biotinylated oligonucleotides were prepared using reagents from Glen Research.…”

Section: Preparation Of Dna Targetsmentioning

confidence: 99%

Transient HMGB protein interactions with B-DNA duplexes and complexes

Zimmerman

Maher

2008

Biochemical and Biophysical Research Communications

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HMGB proteins are abundant, non-histone proteins in eukaryotic chromatin. HMGB proteins contain one or two conserved "HMG boxes" and can be sequence specific or nonspecific in their DNA binding. HMGB proteins cause strong DNA bending and bind preferentially to deformed DNAs. We wish to understand how HMGB proteins increase the apparent flexibility of non-distorted B-form DNA. We test the hypothesis that HMGB proteins bind transiently, creating an ensemble of distorted DNAs with rapidly-interconverting conformations. We show that binding of B-form DNA by HMGB proteins is both weak and transient under conditions where DNA cyclization is strongly enhanced. We also detect novel complexes in which HMGB proteins simultaneously bind more than one DNA duplex.Double-stranded DNA is among the least flexible biopolymers. The local stiffness of DNA is reflected in its persistence length, P, (~150 bp) [1]. The global flexibility of naked DNA in solution is insufficient to allow the degree of compaction required for packaging within cells, or for deformed structures involved in DNA replication, recombination, and transcriptional control. While some controversy exists concerning the accuracy of predictive models for DNA flexibility over short lengths [2;3;4], it is clear that short lengths of DNA require proteins to induce strongly bent and looped conformations.The apparent physical properties of DNA may differ in vitro and in vivo [5;6]. Activation over short distances in assays of eukaryotic transcription activation in vitro raised the possibility of apparent DNA flexibility enhancement by nuclear factors [7]. Indeed, heat-resistant HMGB proteins in nuclear extracts were found to dramatically enhance the cyclization of short DNA restriction fragments by DNA ligase [8].Eukaryotic HMGB proteins are abundant small non-histone chromosomal proteins [9;10;11; 12] that share one or two copies of an ~80-amino acid HMG box domain. Fig. 1A shows the amino acid alignment of HMG box domains, illustrating the partial conservation of intercalating residues [13]. As shown in Fig. 1B, this domain specifies three α-helices (red) whose L-shaped fold engages one face of DNA while delivering one or two intercalating residues into the minor groove [14;15;16]. Biophysical studies have provided some insights into the thermodynamic basis of DNA binding [17;18;19;20]. HMGB proteins to induce DNA kinking or bending of 22;23;24;25], and bind preferentially to distorted DNA structures [16;21;26]. HMGB proteins may stabilize DNA bending either by *To whom correspondence should be addressed at maher@mayo.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all l...

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“…The EcoRI-PvuII fragment isolated from plasmid pGCAT-C3 and 5Ј end labelled at the EcoRI site was incubated with various amounts of recombinant HMGI protein in 100 l of HMGI binding buffer in the presence or absence of 10 g of nonradioactive, sonicated salmon sperm DNA per ml for 10 min at room temperature. The reaction was carried out in the presence of 2 l of a freshly prepared solution of 20 mM Fe(II)-25 mM EDTA, 2 l of a freshly prepared solution of 200 mM sodium ascorbate, and 2 l of 6% hydrogen peroxide as previously described (5).…”

Section: Methodsmentioning

confidence: 99%

Specific Binding of High-Mobility-Group I (HMGI) Protein and Histone H1 to the Upstream AT-Rich Region of the Murine Beta Interferon Promoter: HMGI Protein Acts as a Potential Antirepressor of the Promoter

Bonnefoy

Bandu

Doly

1999

Molecular and Cellular Biology

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The high-mobility-group I (HMGI) protein is a nonhistone component of active chromatin. In this work, we demonstrate that HMGI protein specifically binds to the AT-rich region of the murine beta interferon (IFN-␤) promoter localized upstream of the murine virus-responsive element (VRE). Contrary to what has been described for the human promoter, HMGI protein did not specifically bind to the VRE of the murine IFN-␤ promoter. Stably transfected promoters carrying mutations on this HMGI binding site displayed delayed virusinduced kinetics of transcription. When integrated into chromatin, the mutated promoter remained repressed and never reached normal transcriptional activity. Such a phenomenon was not observed with transiently transfected promoters upon which chromatin was only partially reconstituted. Using UV footprinting, we show that the upstream AT-rich sequences of the murine IFN-␤ promoter constitute a preferential binding region for histone H1. Transfection with a plasmid carrying scaffold attachment regions as well as incubation with distamycin led to the derepression of the IFN-␤ promoter stably integrated into chromatin. In vitro, HMGI protein was able to displace histone H1 from the upstream AT-rich region of the wild-type promoter but not from the promoter carrying mutations on the upstream high-affinity HMGI binding site. Our results suggest that the binding of histone H1 to the upstream AT-rich region of the promoter might be partly responsible for the constitutive repression of the promoter. The displacement by HMGI protein of histone H1 could help to convert the IFN-␤ promoter from a repressed to an active state.

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DNA-binding Parameters of the HU Protein of Escherichia coli to Cruciform DNA

Cited by 135 publications

References 0 publications

DNA Binding Is Not Sufficient for H-NS-mediated Repression ofproU Expression

DNA Binding Is Not Sufficient for H-NS-mediated Repression ofproU Expression

Transient HMGB protein interactions with B-DNA duplexes and complexes

Specific Binding of High-Mobility-Group I (HMGI) Protein and Histone H1 to the Upstream AT-Rich Region of the Murine Beta Interferon Promoter: HMGI Protein Acts as a Potential Antirepressor of the Promoter

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