Publications by authors named "Valerie Lamour"

28 Publications

  • Page 1 of 1

Exonuclease VII repairs quinolone-induced damage by resolving DNA gyrase cleavage complexes.

Sci Adv 2021 Mar 3;7(10). Epub 2021 Mar 3.

Laboratory of Molecular Pharmacology, Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA.

The widely used quinolone antibiotics act by trapping prokaryotic type IIA topoisomerases, resulting in irreversible topoisomerase cleavage complexes (TOPcc). Whereas the excision repair pathways of TOPcc in eukaryotes have been extensively studied, it is not known whether equivalent repair pathways for prokaryotic TOPcc exist. By combining genetic, biochemical, and molecular biology approaches, we demonstrate that exonuclease VII (ExoVII) excises quinolone-induced trapped DNA gyrase, an essential prokaryotic type IIA topoisomerase. We show that ExoVII repairs trapped type IIA TOPcc and that ExoVII displays tyrosyl nuclease activity for the tyrosyl-DNA linkage on the 5'-DNA overhangs corresponding to trapped type IIA TOPcc. ExoVII-deficient bacteria fail to remove trapped DNA gyrase, consistent with their hypersensitivity to quinolones. We also identify an ExoVII inhibitor that synergizes with the antimicrobial activity of quinolones, including in quinolone-resistant bacterial strains, further demonstrating the functional importance of ExoVII for the repair of type IIA TOPcc.
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http://dx.doi.org/10.1126/sciadv.abe0384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7929499PMC
March 2021

Cryo-EM structure of the complete E. coli DNA gyrase nucleoprotein complex.

Nat Commun 2019 10 30;10(1):4935. Epub 2019 Oct 30.

Department of Integrated Structural Biology, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), 1 Rue Laurent Fries, 67404, Illkirch Cedex, France.

DNA gyrase is an essential enzyme involved in the homeostatic control of DNA supercoiling and the target of successful antibacterial compounds. Despite extensive studies, a detailed architecture of the full-length DNA gyrase from the model organism E. coli is still missing. Herein, we report the complete structure of the E. coli DNA gyrase nucleoprotein complex trapped by the antibiotic gepotidacin, using phase-plate single-particle cryo-electron microscopy. Our data unveil the structural and spatial organization of the functional domains, their connections and the position of the conserved GyrA-box motif. The deconvolution of two states of the DNA-binding/cleavage domain provides a better understanding of the allosteric movements of the enzyme complex. The local atomic resolution in the DNA-bound area reaching up to 3.0 Å enables the identification of the antibiotic density. Altogether, this study paves the way for the cryo-EM determination of gyrase complexes with antibiotics and opens perspectives for targeting conformational intermediates.
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http://dx.doi.org/10.1038/s41467-019-12914-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821735PMC
October 2019

Structural Basis for DNA Gyrase Interaction with Coumermycin A1.

J Med Chem 2019 04 3;62(8):4225-4231. Epub 2019 Apr 3.

Integrated Structural Biology Department, IGBMC, UMR7104 CNRS, U1258 Inserm, University of Strasbourg, Illkirch 67404 , France.

Coumermycin A1 is a natural aminocoumarin that inhibits bacterial DNA gyrase, a member of the GHKL proteins superfamily. We report here the first cocrystal structures of gyrase B bound to coumermycin A1, revealing that one coumermycin A1 molecule traps simultaneously two ATP-binding sites. The inhibited dimers from different species adopt distinct sequence-dependent conformations, alternative to the ATP-bound form. These structures provide a basis for the rational development of coumermycin A1 derivatives for antibiotherapy and biotechnology applications.
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http://dx.doi.org/10.1021/acs.jmedchem.8b01928DOI Listing
April 2019

Publisher Correction: Post-translational modifications in DNA topoisomerase 2α highlight the role of a eukaryote-specific residue in the ATPase domain.

Sci Rep 2018 Jul 10;8(1):10673. Epub 2018 Jul 10.

Université de Strasbourg, CNRS UMR7104, INSERM U1258, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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http://dx.doi.org/10.1038/s41598-018-28936-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6039497PMC
July 2018

Post-translational modifications in DNA topoisomerase 2α highlight the role of a eukaryote-specific residue in the ATPase domain.

Sci Rep 2018 06 18;8(1):9272. Epub 2018 Jun 18.

Université de Strasbourg, CNRS UMR7104, INSERM U1258, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France.

Type 2 DNA topoisomerases (Top2) are critical components of key protein complexes involved in DNA replication, chromosome condensation and segregation, as well as gene transcription. The Top2 were found to be the main targets of anticancer agents, leading to intensive efforts to understand their functional and physiological role as well as their molecular structure. Post-translational modifications have been reported to influence Top2 enzyme activities in particular those of the mammalian Top2α isoform. In this study, we identified phosphorylation, and for the first time, acetylation sites in the human Top2α isoform produced in eukaryotic expression systems. Structural analysis revealed that acetylation sites are clustered on the catalytic domains of the homodimer while phosphorylation sites are located in the C-terminal domain responsible for nuclear localization. Biochemical analysis of the eukaryotic-specific K168 residue in the ATPase domain shows that acetylation affects a key position regulating ATP hydrolysis through the modulation of dimerization. Our findings suggest that acetylation of specific sites involved in the allosteric regulation of human Top2 may provide a mechanism for modulation of its catalytic activity.
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http://dx.doi.org/10.1038/s41598-018-27606-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6006247PMC
June 2018

[French network of combined MD-PhD degree programs].

Med Sci (Paris) 2018 May 13;34(5):462-463. Epub 2018 Jun 13.

École de l'Inserm-Liliane Bettencourt, France.

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http://dx.doi.org/10.1051/medsci/20183405020DOI Listing
May 2018

A User-Friendly DNA Modeling Software for the Interpretation of Cryo-Electron Microscopy Data.

Methods Mol Biol 2017 ;1624:193-210

IGBMC, Integrated Structural Biology Department, UMR7104 CNRS, U964 Inserm, Université de Strasbourg, Illkirch, France.

The structural modeling of a macromolecular machine is like a "Lego" approach that is challenged when blocks, like proteins imported from the Protein Data Bank, are to be assembled with an element adopting a serpentine shape, such as DNA templates. DNA must then be built ex nihilo, but modeling approaches are either not user-friendly or very long and fastidious. In this method chapter we show how to use GraphiteLifeExplorer, a software with a simple graphical user interface that enables the sketching of free forms of DNA, of any length, at the atomic scale, as fast as drawing a line on a sheet of paper. We took as an example the nucleoprotein complex of DNA gyrase, a bacterial topoisomerase whose structure has been determined using cryo-electron microscopy (Cryo-EM). Using GraphiteLifeExplorer, we could model in one go a 155 bp long and twisted DNA duplex that wraps around DNA gyrase in the cryo-EM map, improving the quality and interpretation of the final model compared to the initially published data.
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http://dx.doi.org/10.1007/978-1-4939-7098-8_15DOI Listing
April 2018

A no-stop mutation in MAGEB4 is a possible cause of rare X-linked azoospermia and oligozoospermia in a consanguineous Turkish family.

J Assist Reprod Genet 2017 May 11;34(5):683-694. Epub 2017 Apr 11.

Département Génomique Fonctionnelle et Cancer, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de Santé et de Recherche Médicale (INSERM) U964/Centre National de Recherche Scientifique (CNRS) UMR 7104, Université de Strasbourg, 67404, Illkirch, France.

Purpose: The purpose of this study was to identify mutations that cause non-syndromic male infertility using whole exome sequencing of family cases.

Methods: We recruited a consanguineous Turkish family comprising nine siblings with male triplets; two of the triplets were infertile as well as one younger infertile brother. Whole exome sequencing (WES) performed on two azoospermic brothers identified a mutation in the melanoma antigen family B4 (MAGEB4) gene which was confirmed via Sanger sequencing and then screened for on control groups and unrelated infertile subjects. The effect of the mutation on messenger RNA (mRNA) and protein levels was tested after in vitro cell transfection. Structural features of MAGEB4 were predicted throughout the conserved MAGE domain.

Results: The novel single-base substitution (c.1041A>T) in the X-linked MAGEB4 gene was identified as a no-stop mutation. The mutation is predicted to add 24 amino acids to the C-terminus of MAGEB4. Our functional studies were unable to detect any effect either on mRNA stability, intracellular localization of the protein, or the ability to homodimerize/heterodimerize with other MAGE proteins. We thus hypothesize that these additional amino acids may affect the proper protein interactions with MAGEB4 partners.

Conclusion: The whole exome analysis of a consanguineous Turkish family revealed MAGEB4 as a possible new X-linked cause of inherited male infertility. This study provides the first clue to the physiological function of a MAGE protein.
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http://dx.doi.org/10.1007/s10815-017-0900-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5427651PMC
May 2017

Chemical cross-linking and mass spectrometry to determine the subunit interaction network in a recombinant human SAGA HAT subcomplex.

Protein Sci 2015 Aug 14;24(8):1232-46. Epub 2015 Apr 14.

Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes (LSMIS) UMR 7140 CNRS/Université de Strasbourg - "Chimie de la Matière Complexe", 1 Rue Blaise Pascal, 67008, Strasbourg, France.

Understanding the way how proteins interact with each other to form transient or stable protein complexes is a key aspect in structural biology. In this study, we combined chemical cross-linking with mass spectrometry to determine the binding stoichiometry and map the protein-protein interaction network of a human SAGA HAT subcomplex. MALDI-MS equipped with high mass detection was used to follow the cross-linking reaction using bis[sulfosuccinimidyl] suberate (BS3) and confirm the heterotetrameric stoichiometry of the specific stabilized subcomplex. Cross-linking with isotopically labeled BS3 d0-d4 followed by trypsin digestion allowed the identification of intra- and intercross-linked peptides using two dedicated search engines: pLink and xQuest. The identified interlinked peptides suggest a strong network of interaction between GCN5, ADA2B and ADA3 subunits; SGF29 is interacting with GCN5 and ADA3 but not with ADA2B. These restraint data were combined to molecular modeling and a low-resolution interacting model for the human SAGA HAT subcomplex could be proposed, illustrating the potential of an integrative strategy using cross-linking and mass spectrometry for addressing the structural architecture of multiprotein complexes.
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http://dx.doi.org/10.1002/pro.2676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4534174PMC
August 2015

[Structural insight into negative DNA supercoiling by DNA gyrase, a bacterial type 2A DNA topoisomerase].

Med Sci (Paris) 2014 Dec 24;30(12):1081-4. Epub 2014 Dec 24.

IGBMC, départment de biologie structurale intégrative, CNRS UMR7104, Inserm U964, université de Strasbourg, 1, rue Laurent Fries, 67400 Illkirch, France - hôpitaux universitaires de Strasbourg, fédération de médecine translationnelle de Strasbourg, 67000 Strasbourg, France.

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http://dx.doi.org/10.1051/medsci/20143012009DOI Listing
December 2014

Structural insight into negative DNA supercoiling by DNA gyrase, a bacterial type 2A DNA topoisomerase.

Nucleic Acids Res 2013 Sep 26;41(16):7815-27. Epub 2013 Jun 26.

IGBMC, Integrated Structural Biology Department, UMR7104 CNRS, U964 Inserm, Université de Strasbourg, 67400 Illkirch, France, Institut de Chimie de Strasbourg, Université de Strasbourg, UMR7177 CNRS, 67000 Strasbourg, France and Hôpitaux Universitaires de Strasbourg, 67000 Strasbourg, France.

Type 2A DNA topoisomerases (Topo2A) remodel DNA topology during replication, transcription and chromosome segregation. These multisubunit enzymes catalyze the transport of a double-stranded DNA through a transient break formed in another duplex. The bacterial DNA gyrase, a target for broad-spectrum antibiotics, is the sole Topo2A enzyme able to introduce negative supercoils. We reveal here for the first time the architecture of the full-length Thermus thermophilus DNA gyrase alone and in a cleavage complex with a 155 bp DNA duplex in the presence of the antibiotic ciprofloxacin, using cryo-electron microscopy. The structural organization of the subunits of the full-length DNA gyrase points to a central role of the ATPase domain acting like a 'crossover trap' that may help to sequester the DNA positive crossover before strand passage. Our structural data unveil how DNA is asymmetrically wrapped around the gyrase-specific C-terminal β-pinwheel domains and guided to introduce negative supercoils through cooperativity between the ATPase and β-pinwheel domains. The overall conformation of the drug-induced DNA binding-cleavage complex also suggests that ciprofloxacin traps a DNA pre-transport conformation.
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http://dx.doi.org/10.1093/nar/gkt560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3763546PMC
September 2013

Nondenaturing chemical proteomics for protein complex isolation and identification.

Chembiochem 2010 Nov;11(17):2359-61

Laboratory of Functional Chemo-Systems UMR 7199, 74 Route du Rhin, 67401 Illkirch-Graffenstaden, France.

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http://dx.doi.org/10.1002/cbic.201000574DOI Listing
November 2010

Computational tools in protein crystallography.

Methods Mol Biol 2010 ;673:129-56

National Centre for Biological Sciences, Bangalore, India.

Protein crystallography emerged in the early 1970s and is, to this day, one of the most powerful techniques for the analysis of enzyme mechanisms and macromolecular interactions at the atomic level. It is also an extremely powerful tool for drug design. This field has evolved together with developments in computer science and molecular biology, allowing faster three-dimensional structure determination of complex biological assemblies. In recent times, structural genomics initiatives have pushed the development of methods to further speed up this process. The algorithms initially defined in the last decade for structure determination are now more and more elaborate, but the computational tools have evolved toward simpler and more user-friendly packages and web interfaces. We present here a modest overview of the popular software packages that have been developed for solving protein structures, and give a few guidelines and examples for structure determination using the two most popular methods, molecular replacement and multiple anomalous dispersion.
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http://dx.doi.org/10.1007/978-1-60761-842-3_8DOI Listing
December 2010

Structural basis for the bacterial transcription-repair coupling factor/RNA polymerase interaction.

Nucleic Acids Res 2010 Dec 11;38(22):8357-69. Epub 2010 Aug 11.

Laboratory of Molecular Biophysics, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.

The transcription-repair coupling factor (TRCF, the product of the mfd gene) is a widely conserved bacterial protein that mediates transcription-coupled DNA repair. TRCF uses its ATP-dependent DNA translocase activity to remove transcription complexes stalled at sites of DNA damage, and stimulates repair by recruiting components of the nucleotide excision repair pathway to the site. A protein/protein interaction between TRCF and the β-subunit of RNA polymerase (RNAP) is essential for TRCF function. CarD (also called CdnL), an essential regulator of rRNA transcription in Mycobacterium tuberculosis, shares a homologous RNAP interacting domain with TRCF and also interacts with the RNAP β-subunit. We determined the 2.9-Å resolution X-ray crystal structure of the RNAP interacting domain of TRCF complexed with the RNAP-β1 domain, which harbors the TRCF interaction determinants. The structure reveals details of the TRCF/RNAP protein/protein interface, providing a basis for the design and interpretation of experiments probing TRCF, and by homology CarD, function and interactions with the RNAP.
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http://dx.doi.org/10.1093/nar/gkq692DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3001067PMC
December 2010

A chemical labeling strategy for proteomics under nondenaturing conditions.

Chembiochem 2010 Jan;11(1):79-82

Laboratory of Functional Chemo-Systems UMR 7199, 74 Route du Rhin, Illkirch-Graffenstaden, France.

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http://dx.doi.org/10.1002/cbic.200900641DOI Listing
January 2010

Crystal structure of the in vivo-assembled Bacillus subtilis Spx/RNA polymerase alpha subunit C-terminal domain complex.

J Struct Biol 2009 Nov 4;168(2):352-6. Epub 2009 Jul 4.

The Rockefeller University, New York, NY 10065, USA.

The Bacillus subtilis Spx protein is a global transcription factor that interacts with the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD) and regulates transcription of genes involved in thiol-oxidative stress, sporulation, competence, and organosulfur metabolism. Here we determined the X-ray crystal structure of the Spx/alphaCTD complex from an entirely new crystal form than previously reported [Newberry, K.J., Nakano, S., Zuber, P., Brennan, R.G., 2005. Crystal structure of the Bacillus subtilis anti-alpha, global transcriptional regulator, Spx, in complex with the alpha C-terminal domain of RNA polymerase. Proc. Natl. Acad. Sci. USA 102, 15839-15844]. Comparison of the previously reported sulfate-bound complex and our sulfate-free complex reveals subtle conformational changes that may be important for the role of Spx in regulating organosulfur metabolism.
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http://dx.doi.org/10.1016/j.jsb.2009.07.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2757488PMC
November 2009

How to switch off a histidine kinase: crystal structure of Geobacillus stearothermophilus KinB with the inhibitor Sda.

J Mol Biol 2009 Feb 11;386(1):163-77. Epub 2008 Dec 11.

The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.

Entry to sporulation in bacilli is governed by a histidine kinase phosphorelay, a variation of the predominant signal transduction mechanism in prokaryotes. Sda directly inhibits sporulation histidine kinases in response to DNA damage and replication defects. We determined a 2.0-A-resolution X-ray crystal structure of the intact cytoplasmic catalytic core [comprising the dimerization and histidine phosphotransfer domain (DHp domain), connected to the ATP binding catalytic domain] of the Geobacillus stearothermophilus sporulation kinase KinB complexed with Sda. Structural and biochemical analyses reveal that Sda binds to the base of the DHp domain and prevents molecular transactions with the DHp domain to which it is bound by acting as a simple molecular barricade. Sda acts to sterically block communication between the catalytic domain and the DHp domain, which is required for autophosphorylation, as well as to sterically block communication between the response regulator Spo0F and the DHp domain, which is required for phosphotransfer and phosphatase activities.
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http://dx.doi.org/10.1016/j.jmb.2008.12.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2740816PMC
February 2009

Crystal structure of Escherichia coli Rnk, a new RNA polymerase-interacting protein.

J Mol Biol 2008 Nov 12;383(2):367-79. Epub 2008 Aug 12.

Laboratory of Molecular Biophysics, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.

Sequence-based searches identified a new family of genes in proteobacteria, named rnk, which shares high sequence similarity with the C-terminal domains of the Gre factors (GreA and GreB) and the Thermus/Deinococcus anti-Gre factor Gfh1. We solved the X-ray crystal structure of Escherichia coli regulator of nucleoside kinase (Rnk) at 1.9 A resolution using the anomalous signal from the native protein. The Rnk structure strikingly resembles those of E. coli GreA and GreB and Thermus Gfh1, all of which are RNA polymerase (RNAP) secondary channel effectors and have a C-terminal domain belonging to the FKBP fold. Rnk, however, has a much shorter N-terminal coiled coil. Rnk does not stimulate transcript cleavage in vitro, nor does it reduce the lifetime of the complex formed by RNAP on promoters. We show that Rnk competes with the Gre factors and DksA (another RNAP secondary channel effector) for binding to RNAP in vitro, and although we found that the concentration of Rnk in vivo was much lower than that of DksA, it was similar to that of GreB, consistent with a potential regulatory role for Rnk as an anti-Gre factor.
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http://dx.doi.org/10.1016/j.jmb.2008.08.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2613045PMC
November 2008

Crystal structure of the Escherichia coli regulator of sigma70, Rsd, in complex with sigma70 domain 4.

J Mol Biol 2007 Sep 3;372(3):649-59. Epub 2007 Jul 3.

The Rockefeller University, Box 224, 1230 York Avenue, New York, NY 10065, USA.

The Escherichia coli Rsd protein binds tightly and specifically to the RNA polymerase (RNAP) sigma(70) factor. Rsd plays a role in alternative sigma factor-dependent transcription by biasing the competition between sigma(70) and alternative sigma factors for the available core RNAP. Here, we determined the 2.6 A-resolution X-ray crystal structure of Rsd bound to sigma(70) domain 4 (sigma(70)(4)), the primary determinant for Rsd binding within sigma(70). The structure reveals that Rsd binding interferes with the two primary functions of sigma(70)(4), core RNAP binding and promoter -35 element binding. Interestingly, the most highly conserved Rsd residues form a network of interactions through the middle of the Rsd structure that connect the sigma(70)(4)-binding surface with three cavities exposed on distant surfaces of Rsd, suggesting functional coupling between sigma(70)(4) binding and other binding surfaces of Rsd, either for other proteins or for as yet unknown small molecule effectors. These results provide a structural basis for understanding the role of Rsd, as well as its ortholog, AlgQ, a positive regulator of Pseudomonas aeruginosa virulence, in transcription regulation.
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http://dx.doi.org/10.1016/j.jmb.2007.06.081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2083641PMC
September 2007

Recombinant Thermus aquaticus RNA polymerase for structural studies.

J Mol Biol 2006 May 23;359(1):110-21. Epub 2006 Mar 23.

Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08854, USA.

Advances in the structural biology of bacterial transcription have come from studies of RNA polymerases (RNAPs) from the thermophilic eubacteria Thermus aquaticus (Taq) and Thermus thermophilus (Tth). These structural studies have been limited by the fact that only endogenous Taq or Tth RNAP, laboriously purified from large quantities of Taq or Tth cell paste and offering few options for genetic modification, is suitable for structural studies. Recombinant systems for the preparation of Taq RNAP by co-overexpression and assembly in the heterologous host, Escherichia coli, have been described, but these did not yield enzyme suitable for crystallographic studies. Here we describe recombinant systems for the preparation of Taq RNAP harboring full or partial deletions of the Taq beta' non-conserved domain (NCD), yielding enzyme suitable for crystallographic studies. This opens the way for structural studies of genetically manipulated enzymes, allowing the preparation of more crystallizable enzymes and facilitating detailed structure/function analysis. Characterization of the Taqbeta'NCD deletion mutants generated in this study showed that the beta'NCD is important for the efficient binding of the sigma subunit, confirming previous hypotheses. Finally, preliminary structural analysis (at 4.1Angstroms resolution) of one of the recombinant mutants revealed a previously unobserved conformation of the beta-flap, further defining the range of conformations accessible to this flexible structural element.
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http://dx.doi.org/10.1016/j.jmb.2006.03.009DOI Listing
May 2006

Crystal structure of Thermus aquaticus Gfh1, a Gre-factor paralog that inhibits rather than stimulates transcript cleavage.

J Mol Biol 2006 Feb 17;356(1):179-88. Epub 2005 Nov 17.

The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.

Transcription elongation in bacteria is promoted by Gre-factors, which stimulate an endogenous, endonucleolytic transcript cleavage activity of the RNA polymerase. A GreA paralog, Gfh1, present in Thermus aquaticus and Thermus thermophilus, has the opposite effect on elongation complexes, inhibiting rather than stimulating transcript cleavage. We have determined the 3.3 angstroms-resolution X-ray crystal structure of T.aquaticus Gfh1. The structure reveals an N-terminal and a C-terminal domain with close structural similarity to the domains of GreA, but with an unexpected conformational change in terms of the orientation of the domains with respect to each other. However, structural and functional analysis suggests that when complexed with RNA polymerase, Gfh1 adopts a conformation similar to that of GreA. These results reveal considerable structural flexibility for Gfh1, and for Gre-factors in general, as suggested by structural modeling, and point to a possible role for the conformational switch in Gre-factor and Gfh1 regulation. The opposite functional effect of Gfh1 compared with GreA may be determined by three structural characteristics. First, Gfh1 lacks the basic patch present in Gre-factors that likely plays a role in anchoring the 3'-fragment of the back-tracked RNA. Second, the loop at the tip of the N-terminal coiled-coil is highly flexible and contains extra acidic residues compared with GreA. Third, the N-terminal coiled-coil finger lacks a kink in the first alpha-helix, resulting in a straight coiled-coil compared with GreA. The latter two characteristics suggest that Gfh1 chelates a magnesium ion in the RNA polymerase active site (like GreA) but in a catalytically inactive configuration.
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http://dx.doi.org/10.1016/j.jmb.2005.10.083DOI Listing
February 2006

Domain architecture of the p62 subunit from the human transcription/repair factor TFIIH deduced by limited proteolysis and mass spectrometry analysis.

Biochemistry 2004 Nov;43(45):14420-30

Laboratoire de Biologie et Génomique Structurale, Institut de Génétique et de Biologie Moléculaire et Cellulaire, BP 163, 67404 Illkirch Cedex, France.

TFIIH is a multiprotein complex that plays a central role in both transcription and DNA repair. The subunit p62 is a structural component of the TFIIH core that is known to interact with VP16, p53, Eralpha, and E2F1 in the context of activated transcription, as well as with the endonuclease XPG in DNA repair. We used limited proteolysis experiments coupled to mass spectrometry to define structural domains within the conserved N-terminal part of the molecule. The first domain identified resulted from spontaneous proteolysis and corresponds to residues 1-108. The second domain encompasses residues 186-240, and biophysical characterization by fluorescence studies and NMR analysis indicated that it is at least partially folded and thus may correspond to a structural entity. This module contains a region of high sequence conservation with an invariant FWxxPhiPhi motif (Phi representing either tyrosine or phenylalanine), which was also found in other protein families and could play a key role as a protein-protein recognition module within TFIIH. The approach used in this study is general and can be straightforwardly applied to other multidomain proteins and/or multiprotein assemblies.
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http://dx.doi.org/10.1021/bi048884cDOI Listing
November 2004

Proteolytic cleavage of the hyperthermophilic topoisomerase I from Thermotoga maritima does not impair its enzymatic properties.

Biochim Biophys Acta 2004 Aug;1700(2):161-70

Laboratoire d'Enzymologie des Acides Nucléiques, Institut de Génétique et Microbiologie, UMR 8621 CNRS, Bât. 400, Université de Paris Sud, Centre d'Orsay, 91405 Orsay Cedex, France.

Using limited proteolysis, we show that the hyperthermophilic topoisomerase I from Thermotoga maritima exhibits a unique hot spot susceptible to proteolytic attack with a variety of proteases. The remaining of the protein is resistant to further proteolysis, which suggests a compact folding of the thermophilic topoisomerase, when compared to its mesophilic Escherichia coli homologue. We further show that a truncated version of the T. maritima enzyme, lacking the last C-terminal 93 amino acids is more susceptible to proteolysis, which suggests that the C-terminal region of the topoisomerase may be important to maintain the compact folding of the enzyme. The hot spot of cleavage is located around amino acids 326-330 and probably corresponds to an exposed loop of the protein, near the active site tyrosine in charge of DNA cleavage and religation. Location of this protease sensitive region in the vicinity of bound DNA is consistent with the partial protection observed in the presence of different DNA substrates. Unexpectedly, although proteolysis splits the enzyme in two halves, each containing part of the motifs involved in catalysis, trypsin-digested topoisomerase I retains full DNA binding, cleavage, and relaxation activities, full thermostability and also the same hydrodynamic and spectral properties as undigested samples. This supports the idea that the two fragments which are generated by proteolysis remain correctly folded and tightly associated after proteolytic cleavage.
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http://dx.doi.org/10.1016/j.bbapap.2004.04.009DOI Listing
August 2004

TFIIH contains a PH domain involved in DNA nucleotide excision repair.

Nat Struct Mol Biol 2004 Jul 13;11(7):616-22. Epub 2004 Jun 13.

Institut de Génétique et de Biologie Moléculaire et Cellulaire, UMR 7104, 1 Rue Laurent Fries, BP 10142, 67404 Illkirch Cedex, France.

The human general transcription factor TFIIH is involved in both transcription and DNA repair. We have identified a structural domain in the core subunit of TFIIH, p62, which is absolutely required for DNA repair activity through the nucleotide excision repair pathway. Using coimmunoprecipitation experiments, we showed that this activity involves the interaction between the N-terminal domain of p62 and the 3' endonuclease XPG, a major component of the nucleotide excision repair machinery. Furthermore, we reconstituted a functional TFIIH particle with a mutant of p62 lacking the N-terminal domain, showing that this domain is not required for assembly of the TFIIH complex and basal transcription. We solved its three-dimensional structure and found an unpredicted pleckstrin homology and phosphotyrosine binding (PH/PTB) domain, uncovering a new class of activity for this fold.
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http://dx.doi.org/10.1038/nsmb782DOI Listing
July 2004

ATP-bound conformation of topoisomerase IV: a possible target for quinolones in Streptococcus pneumoniae.

J Bacteriol 2003 Oct;185(20):6137-46

INSERM E0004, Laboratoire de Recherche Moléculaire sur les Antibiotiques, UFR Broussais-Hôtel-Dieu, Université Paris VI, 75270 Paris Cedex 06, France.

Topoisomerase IV, a C(2)E(2) tetramer, is involved in the topological changes of DNA during replication. This enzyme is the target of antibacterial compounds, such as the coumarins, which target the ATP binding site in the ParE subunit, and the quinolones, which bind, outside the active site, to the quinolone resistance-determining region (QRDR). After site-directed and random mutagenesis, we found some mutations in the ATP binding site of ParE near the dimeric interface and outside the QRDR that conferred quinolone resistance to Streptococcus pneumoniae, a bacterial pathogen. Modeling of the N-terminal, 43-kDa ParE domain of S. pneumoniae revealed that the most frequent mutations affected conserved residues, among them His43 and His103, which are involved in the hydrogen bond network supporting ATP hydrolysis, and Met31, at the dimeric interface. All mutants showed a particular phenotype of resistance to fluoroquinolones and an increase in susceptibility to novobiocin. All mutations in ParE resulted in resistance only when associated with a mutation in the QRDR of the GyrA subunit. Our models of the closed and open conformations of the active site indicate that quinolones preferentially target topoisomerase IV of S. pneumoniae in its ATP-bound closed conformation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC225018PMC
http://dx.doi.org/10.1128/jb.185.20.6137-6146.2003DOI Listing
October 2003

p52 Mediates XPB function within the transcription/repair factor TFIIH.

J Biol Chem 2002 Aug 21;277(35):31761-7. Epub 2002 Jun 21.

Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/Universite Louis Pasteur, B. P.10142, 67404 Illkirch Cedex, France.

To further our understanding of the transcription/DNA repair factor TFIIH, we investigated the role of its p52 subunit in TFIIH function. Using a completely reconstituted in vitro transcription or nucleotide excision repair (NER) system, we show that deletion of the C-terminal region of p52 results in a dramatic reduction of TFIIH NER and transcription activities. This mutation prevents promoter opening and has no effect on the other enzymatic activities of TFIIH. Moreover, we demonstrate that intact p52 is needed to anchor the XPB helicase within TFIIH, providing an explanation for the transcription and NER defects observed with the mutant p52. We show that these two subunits physically interact and map domains involved in the interface. Taken together, our results show that the p52/Tfb2 subunit of TFIIH regulates the function of XPB through pair-wise interactions as described previously for p44 and XPD.
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http://dx.doi.org/10.1074/jbc.M203792200DOI Listing
August 2002

DNA gyrase interaction with coumarin-based inhibitors: the role of the hydroxybenzoate isopentenyl moiety and the 5'-methyl group of the noviose.

Biochemistry 2002 Jun;41(23):7217-23

UMR CNRS 6032, UFR de Pharmacie, 27 Bd Jean Moulin, 13385 Marseille Cedex 5, France.

DNA gyrase is a major bacterial protein that is involved in replication and transcription and catalyzes the negative supercoiling of bacterial circular DNA. DNA gyrase is a known target for antibacterial agents since its blocking induces bacterial death. Quinolones, coumarins, and cyclothialidines have been designed to inhibit gyrase. Significant improvements can still be envisioned for a better coumarin-gyrase interaction. In this work, we obtained the crystal costructures of the natural coumarin clorobiocin and a synthetic analogue with the 24 kDa gyrase fragment. We used isothermal titration microcalorimetry and differential scanning calorimetry to obtain the thermodynamic parameters representative of the molecular interactions occurring during the binding process between coumarins and the 24 kDa gyrase fragment. We provide the first experimental evidence that clorobiocin binds gyrase with a stronger affinity than novobiocin. We also demonstrate the crucial role of both the hydroxybenzoate isopentenyl moiety and the 5'-alkyl group on the noviose of the coumarins in the binding affinity for gyrase.
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http://dx.doi.org/10.1021/bi0159837DOI Listing
June 2002

An open conformation of the Thermus thermophilus gyrase B ATP-binding domain.

J Biol Chem 2002 May 15;277(21):18947-53. Epub 2002 Feb 15.

Institut de Génétique et de Biologie Moléculaire, CNRS/INSERM/ULP, BP163, 1 rue Laurent Fries, 67404 Illkirch Cedex, France.

DNA gyrase forms an A(2)B(2) tetramer involved in DNA replication, repair, recombination, and transcription in which the B subunit catalyzes ATP hydrolysis. The Thermus thermophilus and Escherichia coli gyrases are homologues and present the same catalytic activity. When compared with that of the E. coli 43K-5'-adenylyl-beta,gamma-imidodiphosphate complex, the crystal structure of Gyrase B 43K ATPase domain in complex with novobiocin, one of the most potent inhibitors of gyrase shows large conformational changes of the subdomains within the dimer. The stabilization of loop 98-118 closing the active site through dimeric contacts and interaction with domain 2 allows to observe novobiocin-protein interactions that could not be seen in the 24K-inhibitor complexes. Furthermore, this loop adopts a position which defines an "open" conformation of the active site in absence of ATP, in contrast with the "closed" conformation adopted upon ATP binding. All together, these results indicate how the subdomains may propagate conformational changes from the active site and provide crucial information for the design of more specific inhibitors.
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http://dx.doi.org/10.1074/jbc.M111740200DOI Listing
May 2002