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Josette Rouviereyaniv - One of the best experts on this subject based on the ideXlab platform.
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the Hu regulon is composed of genes responding to anaerobiosis acid stress high osmolarity and sos induction
PLOS ONE, 2009Co-Authors: Jacques Oberto, Sabrina Nabti, Valerie Jooste, Herve Mignot, Josette RouviereyanivAbstract:Background The Escherichia coli heterodimeric Hu Protein is a small DNA-bending Protein associated with the bacterial nucleoid. It can introduce negative supercoils into closed circular DNA in the presence of topoisomerase I. Cells lacking Hu grow very poorly and display many phenotypes.
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the Hu regulon is composed of genes responding to anaerobiosis acid stress high osmolarity and sos induction
PLOS ONE, 2009Co-Authors: Jacques Oberto, Sabrina Nabti, Valerie Jooste, Herve Mignot, Josette RouviereyanivAbstract:BACKGROUND: The Escherichia coli heterodimeric Hu Protein is a small DNA-bending Protein associated with the bacterial nucleoid. It can introduce negative supercoils into closed circular DNA in the presence of topoisomerase I. Cells lacking Hu grow very poorly and display many phenotypes. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the transcription profile of every Escherichia coli gene in the absence of one or both Hu subunits. This genome-wide in silico transcriptomic approach, performed in parallel with in vivo genetic experimentation, defined the Hu regulon. This large regulon, which comprises 8% of the genome, is composed of four biologically relevant gene classes whose regulation responds to anaerobiosis, acid stress, high osmolarity, and SOS induction. CONCLUSIONS/SIGNIFICANCE: The regulation a large number of genes encoding enzymes involved in energy metabolism and catabolism pathways by Hu explains the highly pleiotropic phenotype of Hu-deficient cells. The uniform chromosomal distribution of the many operons regulated by Hu strongly suggests that the transcriptional and nucleoid architectural functions of Hu constitute two aspects of a unique Protein-DNA interaction mechanism.
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the binding motif recognized by Hu on both nicked and cruciform dna
The EMBO Journal, 1999Co-Authors: D E Kamashev, A Balandina, Josette RouviereyanivAbstract:The heterodimeric Hu Protein, highly conserved in bacteria and involved in transposition, recombination, DNA repair, etc., shares similarity with histones and HMGs. Hu, which binds DNA with low affinity and without sequence specificity, binds strongly and specifically to DNA junctions and DNA containing single-strand breaks. The fine structure of these specific complexes was studied by footprinting and Hu chemically converted into nucleases. The positioning of Hualphabeta on nicked DNA is asymmetrical and specifically oriented: the beta-arm binds the area surrounding the break whereas the alpha-arm lies on the 3' DNA branch. This positioning necessitates a pronounced bend in the DNA at the discontinuous point, which was estimated by circular permutation assay to be 65 degrees. At junctions, Hu is similarly asymmetrically positioned in an identical orientation: the junction point plays the role of the discontinuous point in the nicked DNA. The Hu binding motif present in both structures is a pair of inclined DNA helices.
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differential binding of the escherichia coli Hu homodimeric forms and heterodimeric form to linear gapped and cruciform dna
Journal of Molecular Biology, 1999Co-Authors: V Pinson, Masayuki Takahashi, Josette RouviereyanivAbstract:Abstract We have shown recently that the relative abundance of the three dimeric forms (α2, αβ and β2) of the Hu Protein from Escherichia coli varies during growth and in response to environmental changes. Using gel retardation assays we have compared the DNA binding properties of the three dimers with different DNA substrates. The determination of their DNA binding parameters shows that the relative affinities of Huαβ and Huα2 are comparable. Both recognize, with a high degree of affinity under stringent conditions, cruciform structures or DNA molecules with a nick or a gap, whereas they bind to linear DNA only at low salt. DNA containing a gap of two nucleotides is in fact the substrate recognized with the highest degree of affinity by these two forms under all conditions. Conversely, Huβ2 binds very poorly to duplex DNA and shows a much lower affinity for nicked or gapped DNAs. However, Huβ2 binds to cruciform DNA structures almost as well as Huαβ and Huα2. This almost exclusive binding of Huβ2 to a unique substrate is surprising in regards of the quasi identity, in the three forms, of the flexible arms considered as the DNA-binding domains of the three forms of Hu. Cruciform DNA may stabilize Huβ2 structure which could be structurally defective.
David Dunlap - One of the best experts on this subject based on the ideXlab platform.
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Protein mediated looping of dna under tension requires supercoiling
Nucleic Acids Research, 2018Co-Authors: Yan Yan, Fenfei Leng, Laura Finzi, David DunlapAbstract:Protein-mediated DNA looping is ubiquitous in chromatin organization and gene regulation, but to what extent supercoiling or nucleoid associated Proteins promote looping is poorly understood. Using the lac repressor (LacI), a paradigmatic loop-mediating Protein, we measured LacI-induced looping as a function of either supercoiling or the concentration of the Hu Protein, an abundant nucleoid Protein in Escherichia coli. Negative supercoiling to physiological levels with magnetic tweezers easily drove the looping probability from 0 to 100% in single DNA molecules under slight tension that likely exists in vivo. In contrast, even saturating (micromolar) concentrations of Hu could not raise the looping probability above 30% in similarly stretched DNA or 80% in DNA without tension. Negative supercoiling is required to induce significant looping of DNA under any appreciable tension.
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Hu Protein and dna supercoiling dramatically enhance lac repressor mediated dna looping
Biophysical Journal, 2016Co-Authors: Fenfei Leng, David Dunlap, Laura FinziAbstract:DNA loops mediated by Proteins that bind with different affinity to distant sites regulate multiple aspects of DNA metabolism, such as transcription, replication and recombination. Binding of accessory Proteins and DNA supercoiling are two factors that affect the formation of DNA loops. Previous studies indicate that the heat unstable Protein (Hu) enhances the formation of short loops perhaps by changing the flexibility of DNA upon binding non-specifically. However, for loops long enough such that DNA stiffness is not limiting, the effect of Hu on loop formation is not well understood. Negative supercoiling also enhances the formation of long loops. To better understand how binding affinity, loop length, Hu and negative supercoiling affect looping, single molecule experiments were performed with the lac repressor Protein (LacI) as a DNA looping Protein. Using tethered particle motion, a titration of the formation of Lac repressor mediated-loop was carried out using three different DNA templates, OID-900-O1, OID-400-O1, and O1-400-O2 (Ostrong-loop size (bp)-Oweak). It showed that long loops are surprisingly efficient across a broad range of concentration. In similar titrations of LacI-induced looping as a function of Hu concentration, Hu greatly enhanced looping in 400 but not the longer 900 bp loops. Magnetic tweezers were used to investigate the effect of supercoiling on the formation of LacI-mediated DNA loops using 2 kbp DNA molecules with a centrally located O1-400-O2. Even slight tension in the magnetic tweezer interfered with looping, however, supercoiling compensated for increased tension. Positive supercoiling enhanced loop formation but less efficiently than negative which increased the looping probability from 0 to 100%. THus the levels of accessory Proteins and supercoiling, two dynamic parameters, enable modulation of regulatory loops the baseline probability of which is established by loop size and binding site affinities.
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the effect of Hu Protein on lac repressor mediated dna looping
Biophysical Journal, 2015Co-Authors: Sandip Kumar, Laura Finzi, David DunlapAbstract:The regulation of transcription includes the formation of DNA loops mediated by Proteins that bind to DNA. DNA stiffness and supercoiling influence DNA loop-formation and can be modified by abundant nucleoid associated-Proteins in bacteria that bind to DNA. Studies indicate substantial redundancy between different nucleoid Proteins in vivo, but deletion of the heat unstable Protein (Hu) makes the formation of short loops between strong recognition sequences dependent on the sequence of the loop segment. The Hu Protein has two subunits (α and β), induces negative supercoiling of DNA, and changes the flexibility of DNA upon binding non-specifically. For loops longer than a persistence length, for which DNA stiffness is not limiting, the effect of Hu on loop formation is not well understood. In tethered particle motion (TPM) experiments, a titration of the formation of a 900 bp loop between strong recognition sequences, OID and O1, as a function of Hu concentration exhibits mild changes across a broad range of concentration (0-900 nM). This was surprising since the overall tether length simultaneously decreased which should have facilitated looping. It suggests that Hu has no specific effect on the lac repressor-induced DNA loop, the effect of Hu is more prominent on shorter loops, or the strong OID operator, which supports up to 90% looping at a variety of loop lengths across a large range of LacI concentrations, overwhelms the effect of Hu. Indeed, this last possibility may be significant, since the formation of a 400 bp loop with wild-type spacing and Lac loop operators, O1 and O2, exhibits a narrower range of looping as a function of LacI concentration with a maximum of 50%.
V Popov - One of the best experts on this subject based on the ideXlab platform.
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enhanced conformational flexibility of the histone like Hu Protein from mycoplasma gallisepticum
Journal of Biomolecular Structure & Dynamics, 2018Co-Authors: D A Altukhov, A A Talyzina, Yulia K Agapova, Anna V Vlaskina, Dmitry A Korzhenevskiy, Eduard V Bocharov, T V Rakitina, V I Timofeev, V PopovAbstract:The histone-like (Hu) Protein is one of the major nucleoid-associated Proteins involved in DNA supercoiling and compaction into bacterial nucleoid as well as in all DNA-dependent transactions. This...
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structural basis of the high thermal stability of the histone like Hu Protein from the mollicute spiroplasma melliferum kc3
Scientific Reports, 2016Co-Authors: Konstantin M Boyko, Anna V Vlaskina, Dmitry A Korzhenevskiy, T V Rakitina, V Popov, Dmitry Kamashev, Y K Agapova, Sergey Yu KleymenovAbstract:The three-dimensional structure of the histone-like Hu Protein from the mycoplasma Spiroplasma melliferum KC3 (HuSpm) was determined at 1.4 A resolution, and the thermal stability of the Protein was evaluated by differential scanning calorimetry. A detailed analysis revealed that the three-dimensional structure of the HuSpm dimer is similar to that of its bacterial homologues but is characterized by stronger hydrophobic interactions at the dimer interface. This HuSpm dimer interface lacks salt bridges but is stabilized by a larger number of hydrogen bonds. According to the DSC data, HuSpm has a high denaturation temperature, comparable to that of Hu Proteins from thermophilic bacteria. To elucidate the structural basis of HuSpm thermal stability, we identified amino acid residues potentially responsible for this property and modified them by site-directed mutagenesis. A comparative analysis of the melting curves of mutant and wild-type HuSpm revealed the motifs that play a key role in Protein thermal stability: non-conserved phenylalanine residues in the hydrophobic core, an additional hydrophobic loop at the N-terminal region of the Protein, the absence of the internal cavity present at the dimer interface of some Hu Proteins, and the presence of additional hydrogen bonds between the monomers that are missing in homologous Proteins.
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expression purification crystallization and preliminary x ray crystallographic analysis of the histone like Hu Protein from spiroplasma melliferum kc3
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2015Co-Authors: Konstantin M Boyko, Dmitry A Korzhenevskiy, T V Rakitina, M A Gorbacheva, Anna Vanyushkina, Dmitry Kamashev, A V Lipkin, V PopovAbstract:Hu Proteins belong to the nucleoid-associated Proteins (NAPs) that are involved in vital processes such as DNA compaction and reparation, gene transcription etc. No data are available on the structures of Hu Proteins from mycoplasmas. To this end, the Hu Protein from the parasitic mycoplasma Spiroplasma melliferum KC3 was cloned, overexpressed in Escherichia coli and purified to homogeneity. Prismatic crystals of the Protein were obtained by the vapour-diffusion technique at 4°C. The crystals diffracted to 1.36 A resolution (the best resolution ever obtained for a Hu Protein). The diffraction data were indexed in space group C2 and the structure of the Protein was solved by the molecular-replacement method with one monomer per asymmetric unit.
Laura Finzi - One of the best experts on this subject based on the ideXlab platform.
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Protein mediated looping of dna under tension requires supercoiling
Nucleic Acids Research, 2018Co-Authors: Yan Yan, Fenfei Leng, Laura Finzi, David DunlapAbstract:Protein-mediated DNA looping is ubiquitous in chromatin organization and gene regulation, but to what extent supercoiling or nucleoid associated Proteins promote looping is poorly understood. Using the lac repressor (LacI), a paradigmatic loop-mediating Protein, we measured LacI-induced looping as a function of either supercoiling or the concentration of the Hu Protein, an abundant nucleoid Protein in Escherichia coli. Negative supercoiling to physiological levels with magnetic tweezers easily drove the looping probability from 0 to 100% in single DNA molecules under slight tension that likely exists in vivo. In contrast, even saturating (micromolar) concentrations of Hu could not raise the looping probability above 30% in similarly stretched DNA or 80% in DNA without tension. Negative supercoiling is required to induce significant looping of DNA under any appreciable tension.
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Hu Protein and dna supercoiling dramatically enhance lac repressor mediated dna looping
Biophysical Journal, 2016Co-Authors: Fenfei Leng, David Dunlap, Laura FinziAbstract:DNA loops mediated by Proteins that bind with different affinity to distant sites regulate multiple aspects of DNA metabolism, such as transcription, replication and recombination. Binding of accessory Proteins and DNA supercoiling are two factors that affect the formation of DNA loops. Previous studies indicate that the heat unstable Protein (Hu) enhances the formation of short loops perhaps by changing the flexibility of DNA upon binding non-specifically. However, for loops long enough such that DNA stiffness is not limiting, the effect of Hu on loop formation is not well understood. Negative supercoiling also enhances the formation of long loops. To better understand how binding affinity, loop length, Hu and negative supercoiling affect looping, single molecule experiments were performed with the lac repressor Protein (LacI) as a DNA looping Protein. Using tethered particle motion, a titration of the formation of Lac repressor mediated-loop was carried out using three different DNA templates, OID-900-O1, OID-400-O1, and O1-400-O2 (Ostrong-loop size (bp)-Oweak). It showed that long loops are surprisingly efficient across a broad range of concentration. In similar titrations of LacI-induced looping as a function of Hu concentration, Hu greatly enhanced looping in 400 but not the longer 900 bp loops. Magnetic tweezers were used to investigate the effect of supercoiling on the formation of LacI-mediated DNA loops using 2 kbp DNA molecules with a centrally located O1-400-O2. Even slight tension in the magnetic tweezer interfered with looping, however, supercoiling compensated for increased tension. Positive supercoiling enhanced loop formation but less efficiently than negative which increased the looping probability from 0 to 100%. THus the levels of accessory Proteins and supercoiling, two dynamic parameters, enable modulation of regulatory loops the baseline probability of which is established by loop size and binding site affinities.
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the effect of Hu Protein on lac repressor mediated dna looping
Biophysical Journal, 2015Co-Authors: Sandip Kumar, Laura Finzi, David DunlapAbstract:The regulation of transcription includes the formation of DNA loops mediated by Proteins that bind to DNA. DNA stiffness and supercoiling influence DNA loop-formation and can be modified by abundant nucleoid associated-Proteins in bacteria that bind to DNA. Studies indicate substantial redundancy between different nucleoid Proteins in vivo, but deletion of the heat unstable Protein (Hu) makes the formation of short loops between strong recognition sequences dependent on the sequence of the loop segment. The Hu Protein has two subunits (α and β), induces negative supercoiling of DNA, and changes the flexibility of DNA upon binding non-specifically. For loops longer than a persistence length, for which DNA stiffness is not limiting, the effect of Hu on loop formation is not well understood. In tethered particle motion (TPM) experiments, a titration of the formation of a 900 bp loop between strong recognition sequences, OID and O1, as a function of Hu concentration exhibits mild changes across a broad range of concentration (0-900 nM). This was surprising since the overall tether length simultaneously decreased which should have facilitated looping. It suggests that Hu has no specific effect on the lac repressor-induced DNA loop, the effect of Hu is more prominent on shorter loops, or the strong OID operator, which supports up to 90% looping at a variety of loop lengths across a large range of LacI concentrations, overwhelms the effect of Hu. Indeed, this last possibility may be significant, since the formation of a 400 bp loop with wild-type spacing and Lac loop operators, O1 and O2, exhibits a narrower range of looping as a function of LacI concentration with a maximum of 50%.
T V Rakitina - One of the best experts on this subject based on the ideXlab platform.
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inhibitor targeting the interface between monomers of Hu Protein from spiroplasma melliferum disrupts conformational dynamics and dna binding properties of the Protein
Crystallography Reports, 2020Co-Authors: Yu K Agapova, D A Altukhov, T V Rakitina, V I Timofeev, D E Kamashev, E V SmirnovaAbstract:Histone-like Hu Proteins are global regulators of the topology of the bacterial genome and are involved in transcriptional regulation. These Proteins are essential for the survival of the simplest free-living microorganisms of the Mollicutes class and a number of pathogenic bacteria, including Mycobacterium tuberculosis and Streptococcus pneumonia, and are promising targets for the design of new antibiotics. However, the competitive inhibition of the DNA-binding ability of Hu Proteins is inefficient because of high flexibility and the large size of the DNA-binding site. Hence, an alternative approach was employed based on searching for molecules targeting the interface between two monomers. The three-dimensional structure of the Hu Protein from Spiroplasma melliferum (HuSpm) was used as a target for the virtual screening of the compound library and for the modeling of HuSpm in complex with the inhibitor. An analysis of the molecular dynamics trajectory calculated for the HuSpm–inhibitor complex confirmed the fact that the binding of the inhibitor at the interface between monomers leads to the disruption of conformational dynamics in the region of the DNA-binding domain. The gel retardation assay showed that the inhibitor actually disturbs DNA-binding activity of the Protein, thereby indicating that this is a promising approach.
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structural plasticity and thermal stability of the histone like Protein from spiroplasma melliferum are due to phenylalanine insertions into the conservative scaffold
Journal of Biomolecular Structure & Dynamics, 2018Co-Authors: V I Timofeev, D A Altukhov, A A Talyzina, Yulia K Agapova, Anna V Vlaskina, Dmitry A Korzhenevskiy, Eduard V Bocharov, Sergey Yu Kleymenov, T V RakitinaAbstract:The histone-like (Hu) Protein is one of the major nucleoid-associated Proteins of the bacterial nucleoid, which shares high sequence and structural similarity with IHF but differs from the latter i...
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enhanced conformational flexibility of the histone like Hu Protein from mycoplasma gallisepticum
Journal of Biomolecular Structure & Dynamics, 2018Co-Authors: D A Altukhov, A A Talyzina, Yulia K Agapova, Anna V Vlaskina, Dmitry A Korzhenevskiy, Eduard V Bocharov, T V Rakitina, V I Timofeev, V PopovAbstract:The histone-like (Hu) Protein is one of the major nucleoid-associated Proteins involved in DNA supercoiling and compaction into bacterial nucleoid as well as in all DNA-dependent transactions. This...
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structural basis of the high thermal stability of the histone like Hu Protein from the mollicute spiroplasma melliferum kc3
Scientific Reports, 2016Co-Authors: Konstantin M Boyko, Anna V Vlaskina, Dmitry A Korzhenevskiy, T V Rakitina, V Popov, Dmitry Kamashev, Y K Agapova, Sergey Yu KleymenovAbstract:The three-dimensional structure of the histone-like Hu Protein from the mycoplasma Spiroplasma melliferum KC3 (HuSpm) was determined at 1.4 A resolution, and the thermal stability of the Protein was evaluated by differential scanning calorimetry. A detailed analysis revealed that the three-dimensional structure of the HuSpm dimer is similar to that of its bacterial homologues but is characterized by stronger hydrophobic interactions at the dimer interface. This HuSpm dimer interface lacks salt bridges but is stabilized by a larger number of hydrogen bonds. According to the DSC data, HuSpm has a high denaturation temperature, comparable to that of Hu Proteins from thermophilic bacteria. To elucidate the structural basis of HuSpm thermal stability, we identified amino acid residues potentially responsible for this property and modified them by site-directed mutagenesis. A comparative analysis of the melting curves of mutant and wild-type HuSpm revealed the motifs that play a key role in Protein thermal stability: non-conserved phenylalanine residues in the hydrophobic core, an additional hydrophobic loop at the N-terminal region of the Protein, the absence of the internal cavity present at the dimer interface of some Hu Proteins, and the presence of additional hydrogen bonds between the monomers that are missing in homologous Proteins.
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expression purification crystallization and preliminary x ray crystallographic analysis of the histone like Hu Protein from spiroplasma melliferum kc3
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2015Co-Authors: Konstantin M Boyko, Dmitry A Korzhenevskiy, T V Rakitina, M A Gorbacheva, Anna Vanyushkina, Dmitry Kamashev, A V Lipkin, V PopovAbstract:Hu Proteins belong to the nucleoid-associated Proteins (NAPs) that are involved in vital processes such as DNA compaction and reparation, gene transcription etc. No data are available on the structures of Hu Proteins from mycoplasmas. To this end, the Hu Protein from the parasitic mycoplasma Spiroplasma melliferum KC3 was cloned, overexpressed in Escherichia coli and purified to homogeneity. Prismatic crystals of the Protein were obtained by the vapour-diffusion technique at 4°C. The crystals diffracted to 1.36 A resolution (the best resolution ever obtained for a Hu Protein). The diffraction data were indexed in space group C2 and the structure of the Protein was solved by the molecular-replacement method with one monomer per asymmetric unit.