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Midonet C.,CNRS Institute of Integrative Biology | Das B.,Translational Health Science and Technology Institute | Sherratt D.J.,University of Oxford
Proceedings of the National Academy of Sciences of the United States of America | Year: 2014

As in most bacteria, topological problems arising from the circularity of the two Vibrio cholerae chromosomes, chrI and chrII, are resolved by the addition of a crossover at a specific site of each chromosome, dif, by two tyrosine recombinases, XerC and XerD. The reaction is under the control of a cell division protein, FtsK, which activates the formation of a Holliday Junction (HJ) intermediate by XerD catalysis that is resolved into product by XerC catalysis. Many plasmids and phages exploit Xer recombination for dimer resolution and for integration, respectively. In all cases so far described, they rely on an alternative recombination pathway in which XerC catalyzes the formation of a HJ independently of FtsK. This is notably the case for CTXφ, the cholera toxin phage. Here, we show that in contrast, integration of TLCφ, a toxin-linked cryptic satellite phage that is almost always found integrated at the chrI dif site before CTXφ, depends on the formation of a HJ by XerD catalysis, which is then resolved by XerC catalysis. The reaction nevertheless escapes the normal cellular control exerted by FtsK on XerD. In addition, we show that the same reaction promotes the excision of TLCφ, along with any CTXφ copy present between dif and its left attachment site, providing a plausible mechanism for how chrI CTXφ copies can be eliminated, as occurred in the second wave of the current cholera pandemic.


Leverenz R.L.,Michigan State University | Sutter M.,Michigan State University | Sutter M.,Lawrence Berkeley National Laboratory | Wilson A.,CEA Saclay Nuclear Research Center | And 14 more authors.
Science | Year: 2015

Pigment-protein and pigment-pigment interactions are of fundamental importance to the light-harvesting and photoprotective functions essential to oxygenic photosynthesis. The orange carotenoid protein (OCP) functions as both a sensor of light and effector of photoprotective energy dissipation in cyanobacteria. We report the atomic-resolution structure of an active form of the OCP consisting of the N-terminal domain and a single noncovalently bound carotenoid pigment. The crystal structure, combined with additional solution-state structural data, reveals that OCP photoactivation is accompanied by a 12 angstrom translocation of the pigment within the protein and a reconfiguration of carotenoid-protein interactions. Our results identify the origin of the photochromic changes in the OCP triggered by light and reveal the structural determinants required for interaction with the light-harvesting antenna during photoprotection. © 2015, American Association for the Advancement of Science. All rights reserved.


Pilotto S.,University of Pavia | Speranzini V.,University of Pavia | Tortorici M.,University of Pavia | Tortorici M.,Institute of Cancer Research | And 10 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2015

With its noncatalytic domains, DNA-binding regions, and a catalytic core targeting the histone tails, LSD1-CoREST (lysine-specific demethylase 1; REST corepressor) is an ideal model system to study the interplay between DNA binding and histone modification in nucleosome recognition. To this end, we covalently associated LSD1-CoREST to semisynthetic nucleosomal particles. This enabled biochemical and biophysical characterizations of nucleosome binding and structural elucidation by small-angle X-ray scattering, which was extensively validated through binding assays and site-directed mutagenesis of functional interfaces. Our results suggest that LSD1-CoREST functions as an ergonomic clamp that induces the detachment of the H3 histone tail from the nucleosomal DNA to make it available for capture by the enzyme active site. The key notion emerging from these studies is the inherently competitive nature of the binding interactions because nucleosome tails, chromatin modifiers, transcription factors, and DNA represent sites for multiple and often mutually exclusive interactions.


Theillet F.-X.,Leibniz Institute for Molecular Pharmacology | Theillet F.-X.,CNRS Institute of Integrative Biology | Binolfi A.,Leibniz Institute for Molecular Pharmacology | Binolfi A.,Max Planck Institute of Biophysics | And 9 more authors.
Nature | Year: 2016

Intracellular aggregation of the human amyloid protein α-synuclein is causally linked to Parkinson's disease. While the isolated protein is intrinsically disordered, its native structure in mammalian cells is not known. Here we use nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy to derive atomic-resolution insights into the structure and dynamics of α-synuclein in different mammalian cell types. We show that the disordered nature of monomeric α-synuclein is stably preserved in non-neuronal and neuronal cells. Under physiological cell conditions, α-synuclein is amino-terminally acetylated and adopts conformations that are more compact than when in buffer, with residues of the aggregation-prone non-amyloid-β component (NAC) region shielded from exposure to the cytoplasm, which presumably counteracts spontaneous aggregation. These results establish that different types of crowded intracellular environments do not inherently promote α-synuclein oligomerization and, more generally, that intrinsic structural disorder is sustainable in mammalian cells. © 2016 Macmillan Publishers Limited. All rights reserved.


Valens M.,CNRS Institute of Integrative Biology | Thiel A.,CNRS Institute of Integrative Biology | Boccard F.,CNRS Institute of Integrative Biology
PLoS Genetics | Year: 2016

The Ori region of bacterial genomes is segregated early in the replication cycle of bacterial chromosomes. Consequently, Ori region positioning plays a pivotal role in chromosome dynamics. The Ori region of the E. coli chromosome is organized as a macrodomain with specific properties concerning DNA mobility, segregation of loci and long distance DNA interactions. Here, by using strains with chromosome rearrangements and DNA mobility as a read-out, we have identified the MaoP/maoS system responsible for constraining DNA mobility in the Ori region and limiting long distance DNA interactions with other regions of the chromosome. MaoP belongs to a group of proteins conserved in the Enterobacteria that coevolved with Dam methylase including SeqA, MukBEF and MatP that are all involved in the control of chromosome conformation and segregation. Analysis of DNA rings excised from the chromosome demonstrated that the single maoS site is required in cis on the chromosome to exert its effect while MaoP can act both in cis and in trans. The position of markers in the Ori region was affected by inactivating maoP. However, the MaoP/maoS system was not sufficient for positioning the Ori region at the ¼–¾ regions of the cell. We also demonstrate that the replication and the resulting expansion of bulk DNA are localized centrally in the cell. Implications of these results for chromosome positioning and segregation in E. coli are discussed. © 2016 Valens et al.


Carlier M.-F.,CNRS Institute of Integrative Biology | Pernier J.,CNRS Institute of Integrative Biology | Montaville P.,CNRS Institute of Integrative Biology | Shekhar S.,CNRS Institute of Integrative Biology | Kuhn S.,CNRS Institute of Integrative Biology
Cellular and Molecular Life Sciences | Year: 2015

Actin cytoskeleton remodeling, which drives changes in cell shape and motility, is orchestrated by a coordinated control of polarized assembly of actin filaments. Signal responsive, membrane-bound protein machineries initiate and regulate polarized growth of actin filaments by mediating transient links with their barbed ends, which elongate from polymerizable actin monomers. The barbed end of an actin filament thus stands out as a hotspot of regulation of filament assembly. It is the target of both soluble and membrane-bound agonists as well as antagonists of filament assembly. Here, we review the molecular mechanisms by which various regulators of actin dynamics bind, synergize or compete at filament barbed ends. Two proteins can compete for the barbed end via a mutually exclusive binding scheme. Alternatively, two regulators acting individually at barbed ends may be bound together transiently to terminal actin subunits at barbed ends, leading to the displacement of one by the other. The kinetics of these reactions is a key in understanding how filament length and membrane-filament linkage are controlled. It is also essential for understanding how force is produced to shape membranes by mechano-sensitive, processive barbed end tracking machineries like formins and by WASP-Arp2/3 branched filament arrays. A combination of biochemical and biophysical approaches, including bulk solution assembly measurements using pyrenyl-actin fluorescence, single filament dynamics, single molecule fluorescence imaging and reconstituted self-organized filament assemblies, have provided mechanistic insight into the role of actin polymerization in motile processes. © 2015 The Author(s).


Un S.,CNRS Institute of Integrative Biology | Bruch E.M.,CNRS Institute of Integrative Biology
Inorganic Chemistry | Year: 2015

Manganous phosphates have been postulated to play an important role in cells as antioxidants. In situ Mn(II) electron-nuclear double resonance (ENDOR) spectroscopy has been used to measure their speciation in cells. The analyses of such ENDOR spectra and the quantification of cellular Mn(II) phosphates has been based on comparisons to in vitro model complexes and heuristic modeling. In order to put such analyses on a more physical and theoretical footing, the Mn(II)-31P hyperfine interactions of various Mn(II) phosphate complexes have been measured by 95 GHz ENDOR spectroscopy. The dipolar components of these interactions remained relatively constant as a function of pH, esterification, and phosphate chain length, while the isotropic contributions were significantly affected. Counterintuitively, although the manganese-phosphate bonds are weakened by protonation and esterification, they lead to larger isotropic values, indicating higher unpaired-electron spin densities at the phosphorus nuclei. By comparison, extending the phosphate chain with additional phosphate groups lowers the spin density. Density functional theory calculations of model complexes quantitatively reproduced the measured hyperfine couplings and provided detailed insights into how bonding in Mn(II) phosphate complexes modulates the electron-spin polarization and consequently their isotropic hyperfine couplings. These results show that various classes of phosphates can be identified by their ENDOR spectra and provide a theoretical framework for understanding the in situ 31P ENDOR spectra of cellular Mn(II) complexes. © 2015 American Chemical Society.


The natural history of tuberculosis may be tackled by various means, among which the record of molecular scars that have been registered by the Mycobacterium tuberculosis complex (MTBC) genomes transmitted from patient to patient for tens of thousands years and possibly more. Recently discovered polymorphic loci, the CRISPR sequences, are indirect witnesses of the historical phage-bacteria struggle, and may be related to the time when the ancestor of today's tubercle bacilli were environmental bacteria, i.e. before becoming intracellular parasites. In this article, we present what are CRISPRs and try to summarize almost 20 years of research results obtained using the genetic diversity of the CRISPR loci in MTBC as a perspective for studying new models. We show that the study of the diversity of CRISPR sequences, thanks to «spoligotyping», has played a great role in our global understanding of the population structure of MTBC. © 2015 Elsevier Ltd. All rights reserved.


Bridier-Nahmias A.,University Paris Diderot | Bridier-Nahmias A.,French National Conservatory of Arts and Crafts | Tchalikian-Cosson A.,University Paris Diderot | Baller J.A.,University of Minnesota | And 9 more authors.
Science | Year: 2015

Mobile genetic elements are ubiquitous. Their integration site influences genome stability and gene expression. The Ty1 retrotransposon of the yeast Saccharomyces cerevisiae integrates upstream of RNA polymerase III (Pol III)-transcribed genes, yet the primary determinant of target specificity has remained elusive. Here we describe an interaction between Ty1 integrase and the AC40 subunit of Pol III and demonstrate that AC40 is the predominant determinant targeting Ty1 integration upstream of Pol III-transcribed genes. Lack of an integrase-AC40 interaction dramatically alters target site choice, leading to a redistribution of Ty1 insertions in the genome, mainly to chromosome ends. The mechanism of target specificity allows Ty1 to proliferate and yet minimizes genetic damage to its host.


Boussac A.,CNRS Institute of Integrative Biology | Rutherford A.W.,Imperial College London | Sugiura M.,Ehime University | Sugiura M.,Japan Science and Technology Agency
Biochimica et Biophysica Acta - Bioenergetics | Year: 2015

The site for water oxidation in Photosystem II (PSII) goes through five sequential oxidation states (S0 to S4) before O2 is evolved. It consists of a Mn4CaO5-cluster close to a redox-active tyrosine residue (YZ). Cl- is also required for enzyme activity. By using EPR spectroscopy it has been shown that both Ca2+/Sr2+ exchange and Cl-/I- exchange perturb the proportions of centers showing high (S = 5/2) and low spin (S = 1/2) forms of the S2-state. The S3-state was also found to be heterogeneous with: i) a S = 3 form that is detectable by EPR and not sensitive to near-infrared light; and ii) a form that is not EPR visible but in which Mn photochemistry occurs resulting in the formation of a (S2YZ)′ split EPR signal upon near-infrared illumination. In Sr/Cl-PSII, the high spin (S = 5/2) form of S2 shows a marked heterogeneity with a g = 4.3 form generated at low temperature that converts to a relaxed form at g = 4.9 at higher temperatures. The high spin g = 4.9 form can then progress to the EPR detectable form of S3 at temperatures as low as 180 K whereas the low spin (S = 1/2) S2-state can only advance to the S3 state at temperatures 235 K. Both of the two S2 configurations and the two S3 configurations are each shown to be in equilibrium at 235 K but not at 198 K. Since both S2 configurations are formed at 198 K, they likely arise from two specific populations of S1. The existence of heterogeneous populations in S1, S2 and S3 states may be related to the structural flexibility associated with the positioning of the oxygen O5 within the cluster highlighted in computational approaches and which has been linked to substrate exchange. These data are discussed in the context of recent in silico studies of the electron transfer pathways between the S2-state(s) and the S3-state(s). ©2015 Elsevier B.V. All rights reserved.

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