DESCRIPTIF DU TRAVAIL DE RECHERCHE
La vie de tout organisme et sa pérennité de génération en génération dépendent de la stabilité de l’information génétique contenue dans la double-hélice de son ADN et de la dynamique « intrinsèque » ou « extrinsèque » de cette même molécule d’ADN.
De par sa nature hautement électrophile, la molécule d’ADN peut-être en permanence modifiée par des agents chimiques (alkylation, oxydation, etc.) ou physiques (température, radiations UV et gamma).
Dans de nombreux cas, ces modifications de la structure de l’ADN sont associées à des mécanismes de mort cellulaire et de mutagenèse. Ils participent ainsi aux processus de carcinogénèse et de vieillissement. Pour pallier les effets délétères des modifications chimiques de la molécule d’ADN, les organismes vivants ont développé de nombreux mécanismes de réparation de l’ADN qui ont été, dans leurs principes de base, conservés de la bactérie jusqu’à l’Homme.
De plus, tous les processus impliqués dans le métabolisme de l’ADN (réplication, transcription, recombinaison et réparation) sont eux-mêmes associés à une dynamique structurale de l’ADN qui fait intervenir un grand nombre de protéines susceptibles de modifier sa structure. On parle ici de protéines de structure de l’ADN ou « ADN chaperones ».
Dans ce cadre, l’équipe s’intéresse aux bases moléculaires de la reconnaissance et de l’élimination des bases oxydées de l’ADN par les ADN glycosylases du système de réparation par excision de base. Nous observons également la façon dont la structuration de l’ADN par les ADN chaperones peut moduler l’efficacité de ces systèmes.
→ Réparation de l’ADN
L’objectif principal que nous poursuivons sur ce thème repose sur la compréhension, au niveau moléculaire, de la manière dont les ADN glycosylases telles que Fpg (bactérie) et hOgg1 (Homme) sont capables de détecter, reconnaître et exciser les purines oxydées telles que la 8-oxoguanine (mutagène) et les résidus Fapy (létaux), parmi un excès de bases normales.
Nous développons en particulier une approche structurale par cristallographie de complexes enzyme/ADN endommagés. Par cette approche, nous souhaitons concevoir des inhibiteurs sélectifs pour ces enzymes utilisables pour des stratégies anticancéreuses.
→ ADN chaperones
Nous tentons d’appréhender l’implication des ADN chaperones dans la dynamique structurale de l’ADN bactérien et mitochondrial à travers l’étude de l’interaction avec l’ADN (linéaires, circulaires relaxés et surenroulés) des protéines MC1 (archéobactérie), HU (bactérie) et Abf2/Tfam (mitochondrie).
Nous observons également le processus selon lequel ces protéines peuvent moduler l’accessibilité des purines oxydées aux ADN glycosylases telles que Fpg et hOgg1.
Jacob L., Sawma P., Garnier N., Meyer L.A., Fritz J., Hussenet T., Spenlé C., Goetz J., Vermot J., Fernandez A., Baumlin N., Aci-Sèche S., Orend G., Roussel G., Crémel G., Genest M., Hubert P. and Bagnard D.
Inhibition of PlexA1-mediated brain tumor growth and tumor-associated angiogenesis using a transmembrane domain targeting peptide.(2016) Oncotarget, 7 (36) 57851-57865.
Le Meur R., Culard F., Nadan V., Goffinont S., Coste F., Guerin M., Loth K., Landon C. and Castaing B.
The nucleoid-associated protein HU enhances 8-oxoguanine base excision by the formamidopyrimidine-DNA glycosylase. (2015) Biochemical Journal, 471, 13-23.
Biela A., Coste F., Culard F., Guerin M., Goffinont S., Gasteiger K., Cieśla J., Winczura A., Kazimierczuk Z., Gasparutto D., Carell T., Tudek B. and Castaing B.
Zinc finger oxidation of Fpg/Nei DNA glycosylases by 2-thioxanthine : biochemical and X-ray structural characterization. (2014) Nucleic Acids Research, 42, 10748-61.
Gueneau E., Dherin C., Legrand P., Tellier-Lebegue C., Gilquin B., Bonnesoeur P., Londino F., Quemener C., Le Du M.-H., Márquez J. A., Moutiez M., Gondry M., Boiteux S. and Charbonnier J.-B.
Structure of the MutLα C-terminal domain reveals how Mlh1 contributes to Pms1 endonuclease site. (2013) Nature Structural and Molecular Biology, 20, 461-68.
Le Bihan Y. V., Izquierdo M. A., Coste F., Aller P., Culard F., Gehrke T. H., Essalhi K., Carell T. and Castaing B.
5-Hydroxy-5-methylhydantoin DNA containing lesion : a molecular trap for DNA glycosylases. (2011) Nucleic Acids Research, 39, 6277-90.
Membres du groupe thématique
Jacob, L. Sawma, P. Garnier, N. Meyer, L. A. Fritz, J. Hussenet, T. Spenle, C. Goetz, J. Vermot, J. Fernandez, A. Baumlin, N. Aci-Seche, S. Orend, G. Roussel, G. Cremel, G. Genest, M. Hubert, P. Bagnard, D. (2016)
Inhibition of PlexA1-mediated brain tumor growth and tumor-associated angiogenesis using a transmembrane domain targeting peptideOncotarget (2016) 7 (36) 57851-57865 - doi : 10.18632/oncotarget.11072
The neuropilin-plexin receptor complex regulates tumor cell migration and proliferation and thus is an interesting therapeutic target. High expression of neuropilin-1 is indeed associated with a bad prognosis in glioma patients. Q-RTPCR and tissue-array analyses showed here that Plexin-A1 is highly expressed in glioblastoma and that the highest level of expression correlates with the worse survival of patients. We next identified a developmental and tumor-associated pro-angiogenic role of Plexin-A1. Hence, by using molecular simulations and a two-hybrid like assay in parallel with biochemical and cellular assays we developed a specific Plexin-A1 peptidic antagonist disrupting transmembrane domain-mediated oligomerization of the receptor and subsequent signaling and functional activity. We found that this peptide exhibits anti-tumor activity in vivo on different human glioblastoma models including glioma cancer stem cells. Thus, screening Plexin-A1 expression and targeting Plexin-A1 in glioblastoma patients exhibit diagnostic and therapeutic value.
Le Meur, R., Culard, F., Nadan, V., Goffinont, S., Coste, F., Guerin, M., Loth, K., Landon, C. and Castaing, B. (2015)
The nucleoid-associated protein HU enhances 8-oxoguanine base excision by the formamidopyrimidine-DNA glycosylaseBiochemical Journal (2015) 471 (1) 13-23 - doi : 10.1042/BJ20150387
The nucleoid-associated protein HU is involved in numerous DNA transactions and thus is essential in DNA maintenance and bacterial survival. The high affinity of HU for SSBs (single-strand breaks) has suggested its involvement in DNA protection, repair and recombination. SSB-containing DNA are major intermediates transiently generated by bifunctional DNA N-glycosylases that initiate the BER (base excision repair) pathway. Enzyme kinetics and DNA-binding experiments demonstrate that HU enhances the 8-oxoguanine-DNA glycosylase activity of Fpg (formamidopyrimidine-DNA glycosylase) by facilitating the release of the enzyme from its final DNA product (one nucleoside gap). We propose that the displacement of Fpg from its end-DNA product by HU is an active mechanism in which HU recognizes the product when it is still bound by Fpg. Through DNA binding, the two proteins interplay to form a transient ternary complex Fpg/DNA/HU which results in the release of Fpg and the molecular entrapment of SSBs by HU. These results support the involvement of HU in BER in vivo.
ATP-dependent motor activity of the transcription termination factor Rho from Mycobacterium tuberculosisNucleic Acids Research (2015) First published online : May 20, 2015 - doi : 10.1093/nar/gkv505
The bacterial transcription termination factor Rho—a ring-shaped molecular motor displaying directional, ATP-dependent RNA helicase/translocase activity—is an interesting therapeutic target. Recently, Rho from Mycobacterium tuberculosis (MtbRho) has been proposed to operate by a mechanism uncoupled from molecular motor action, suggesting that the manner used by Rho to dissociate transcriptional complexes is not conserved throughout the bacterial kingdom. Here, however, we demonstrate that MtbRho is a bona fide molecular motor and directional helicase which requires a catalytic site competent for ATP hydrolysis to disrupt RNA duplexes or transcription elongation complexes. Moreover, we show that idiosyncratic features of the MtbRho enzyme are conferred by a large, hydrophilic insertion in its N-terminal ‘RNA binding’ domain and by a non-canonical R-loop residue in its C-terminal ‘motor’ domain. We also show that the ‘motor’ domain of MtbRho has a low apparent affinity for the Rho inhibitor bicyclomycin, thereby contributing to explain why M. tuberculosis is resistant to this drug. Overall, our findings support that, in spite of adjustments of the Rho motor to specific traits of its hosting bacterium, the basic principles of Rho action are conserved across species and could thus constitute pertinent screening criteria in high-throughput searches of new Rho inhibitors.
Monitoring RNA Unwinding by the Transcription Termination Factor Rho from Mycobacterium tuberculosisIn "RNA Remodeling Proteins" (2015) vol. 1259, chap 18, 293-311 - doi : 10.1007/978-1-4939-2214-7_18
Transcription termination factor Rho is a ring-shaped, homo-hexamieric RNA translocase that dissociates transcription elongation complexes and transcriptional RNA-DNA duplexes (R-loops) in bacteria. The molecular mechanisms underlying these biological functions have been essentially studied with Rho enzymes from Escherichia coli or close Gram-negative relatives. However, phylo-divergent Rho factors may have distinct properties. Here, we describe methods for the preparation and in vitro characterization (ATPase and helicase activities) of the Rho factor from Mycobacterium tuberculosis, a specimen with uncharacteristic molecular and enzymatic features. These methods set the stage for future studies aimed at better defining the diversity of enzymatic properties of Rho across the bacterial kingdom.
Backbone assignment of the three dimers of HU from Escherichia coli at 293 K : EcHUα2, EcHUβ2 and EcHUαβBiomolecular NMR Assignments (2015) 1-5 - doi : 10.1007/s12104-015-9610-6
HU is one of the major nucleoid-associated proteins involved in bacterial chromosome structure and in all DNA-dependent cellular activities. Similarly to eukaryotic histones, this small dimeric basic protein wraps DNA in a non-sequence specific manner, promoting DNA super-structures. In most bacteria, HU is a homodimeric protein encoded by a single gene. However, in enterobacteria such as Escherichia coli, the presence of two genes coding for two peptidic chains, HUα and HUβ, lead to the coexistence of three forms : two homodimers EcHUα2 and EcHUβ2, as well as a heterodimer EcHUαβ. Genetic and biochemical investigation suggest that each EcHU dimer plays a specific physiological role in bacteria. Their relative abundance depends on the environmental conditions and is driven by an essential, yet unknown, fast outstanding chain-exchange mechanism at physiological temperature. Our goal is to understand this fundamental mechanism from a structural and kinetics standpoint using NMR. For this purpose, the first steps are the assignment of each dimer in their native and intermediate states. Here, we report the backbone assignment of each HU dimers from E. coli at 293 K in their native state.
Ziani, W., Maillard, A. P., Petit-Hartlein, I., Garnier, N., Crouzy, S., Girard, E. and Coves, J. (2014)
The X-ray Structure of NccX from Cupriavidus metallidurans 31A Illustrates Potential Dangers of Detergent Solubilization When Generating and Interpreting Crystal Structures of Membrane ProteinsThe Journal of Biological Chemistry (2014) 289 (45) 31130-31172 - doi : 10.1074/jbc.M114.586537
The x-ray structure of NccX, a type II transmembrane metal sensor, from Cupriavidus metallidurans 31A has been determined at a resolution of 3.12 A. This was achieved after solubilization by dodecylphosphocholine and purification in the presence of the detergent. NccX crystal structure did not match the model based on the extensively characterized periplasmic domain of its closest homologue CnrX. Instead, the periplasmic domains of NccX appeared collapsed against the hydrophobic transmembrane segments, leading to an aberrant topology incompatible with membrane insertion. This was explained by a detergent-induced redistribution of the hydrophobic interactions among the transmembrane helices and a pair of hydrophobic patches keeping the periplasmic domains together in the native dimer. Molecular dynamics simulations performed with the full-length protein or with the transmembrane segments were used along with in vivo homodimerization assays (TOXCAT) to evaluate the determinants of the interactions between NccX protomers. Taken as a whole, computational and experimental results are in agreement with the structural model of CnrX where a cradle-shaped periplasmic metal sensor domain is anchored into the inner membrane by two N-terminal helices. In addition, they show that the main determinant of NccX dimerization is the periplasmic soluble domain and that the interaction between transmembrane segments is highly dynamic. The present work introduces a new crystal structure for a transmembrane protein and, in line with previous studies, substantiates the use of complementary theoretical and in vivo investigations to rationalize a three-dimensional structure obtained in non-native conditions.
Zinc finger oxidation of Fpg/Nei DNA glycosylases by 2-thioxanthine : biochemical and X-ray structural characterizationNucleic Acids Research (2014) 42(16) 10748-10761 - doi : 10.1093/nar/gku613
DNA glycosylases from the Fpg/Nei structural superfamily are base excision repair enzymes involved in the removal of a wide variety of mutagen and potentially lethal oxidized purines and pyrimidines. Although involved in genome stability, the recent discovery of synthetic lethal relationships between DNA glycosylases and other pathways highlights the potential of DNA glycosylase inhibitors for future medicinal chemistry development in cancer therapy. By combining biochemical and structural approaches, the physical target of 2-thioxanthine (2TX), an uncompetitive inhibitor of Fpg, was identified. 2TX interacts with the zinc finger (ZnF) DNA binding domain of the enzyme. This explains why the zincless hNEIL1 enzyme is resistant to 2TX. Crystal structures of the enzyme bound to DNA in the presence of 2TX demonstrate that the inhibitor chemically reacts with cysteine thiolates of ZnF and induces the loss of zinc. The molecular mechanism by which 2TX inhibits Fpg may be generalized to all prokaryote and eukaryote ZnF-containing Fpg/Nei-DNA glycosylases. Cell experiments show that 2TX can operate in cellulo on the human Fpg/Nei DNA glycosylases. The atomic elucidation of the determinants for the interaction of 2TX to Fpg provides the foundation for the future design and synthesis of new inhibitors with high efficiency and selectivity.
Interaction of Ddc1 and RPA with single-stranded/double-stranded DNA junctions in yeast whole cell extracts : Proteolytic degradation of the large subunit of replication protein A in ddc1Δ strainsDNA Repair (2014) 22, 30-40 -doi : http://dx.doi.org/10.1016/j.dnarep.2014.07.002
To characterize proteins that interact with single-stranded/double-stranded (ss/ds) DNA junctions in whole cell free extracts of Saccharomyces cerevisiae, we used [32P]-labeled photoreactive partial DNA duplexes containing a 3′-ss/ds-junction (3′-junction) or a 5′-ss/ds-junction (5′-junction). Identification of labeled proteins was achieved by MALDI-TOF mass spectrometry peptide mass fingerprinting and genetic analysis. In wild-type extract, one of the components of the Ddc1-Rad17-Mec3 complex, Ddc1, was found to be preferentially photocrosslinked at a 3′-junction. On the other hand, RPAp70, the large subunit of the replication protein A (RPA), was the predominant crosslinking product at a 5′-junction. Interestingly, ddc1Δ extracts did not display photocrosslinking of RPAp70 at a 5′-junction. The results show that RPAp70 crosslinked to DNA with a 5′-junction is subject to limited proteolysis in ddc1Δ extracts, whereas it is stable in WT, rad17Δ, mec3Δ and mec1Δ extracts. The degradation of the RPAp70-DNA adduct in ddc1Δ extract is strongly reduced in the presence of the proteasome inhibitor MG 132. We also addressed the question of the stability of free RPA, using anti-RPA antibodies. The results show that RPAp70 is also subject to proteolysis without photocrosslinking to DNA upon incubation in ddc1Δ extract. The data point to a novel property of Ddc1, modulating the turnover of DNA binding proteins such as RPAp70 by the proteasome.
Transmembrane domain targeting peptide antagonizing ErbB2/Neu inhibits breast tumor growth and metastasisCell Reports (2014) 8 (6) 1714-1721 - doi : 10.1016/j.celrep.2014.07.044
Breast cancer is still a deadly disease despite major achievements in targeted therapies designed to block ligands or ligand-binding subunits of major tyrosine kinase receptors. Relapse is significant and metastases deleterious, which demands novel strategies for fighting this disease. Here, we report a proof-of-concept experiment demonstrating that small peptides interfering with the transmembrane domain of the tyrosine kinase epidermal growth factor receptor ErbB2 exhibit anticancer properties when used at micromolar dosages in a genetically engineered mouse model of breast cancer. Different assays demonstrate the specificity of the ErbB2-targeting peptide, which induces long-term reduction of ErbB2 phosphorylation and Akt signaling consistent with reduced tumor cell proliferation and increased survival. Microcomputed tomography analysis established the antimetastatic activity of the peptide and its impact on primary tumor growth. This reveals the interior of the cell membrane as an unexplored dimension for drug design.
Transmembrane Recognition of the Semaphorin Co- Receptors Neuropilin 1 and Plexin A1 : Coarse-Grained SimulationsPLoS One (2014) 9 (5) e97779 - doi : doi:10.1371/journal.pone.0097779
The cancer associated class 3 semaphorins require direct binding to neuropilins and association to plexins to trigger cell signaling. Here, we address the role of the transmembrane domains of neuropilin 1 and plexin A1 for the dimerization of the
two receptors by characterizing the assembly in lipid bilayers using coarse-grained molecular dynamics simulations. From experimental evidence using a two-hybrid system showing the biochemical association of the two receptors transmembrane domains, we performed molecular simulations in DOPC and POPC demonstrating spontaneously assembly to form homodimers and heterodimers with a very high propensity for right-handed packing of the helices. Inversely, lefthanded packing was observed with a very low propensity. This mode of packing was observed uniquely when the plexin A1
transmembrane domain was involved in association. Potential of mean force calculations were used to predict a hierarchy of self-association for the monomers : the two neuropilin 1 transmembrane domains strongly associated, neuropilin 1 and plexin A1 transmembrane domains associated less and the two plexin A1 transmembrane domains weakly but significantly associated. We demonstrated that homodimerization and heterodimerization are driven by GxxxG motifs, and that the sequence context modulates the packing mode of the plexin A1 transmembrane domains. This work presents major
advances towards our understanding of membrane signaling platforms assembly through membrane domains and provides exquisite information for the design of antagonist drugs defining a novel class of therapeutic agents.
Bile-mediated activation of the acrAB and tolC multidrug efflux genes occurs mainly through transcriptional derepression of ramA in Salmonella enterica serovar TyphimuriumJournal of Antimicrobial Chemotherapy (2014) [Epub ahead of print] - doi : 10.1093/jac/dku140
OBJECTIVES : In Salmonella Typhimurium, the genes encoding the AcrAB-TolC multidrug efflux system are mainly regulated by the ramRA locus, composed of the divergently transcribed ramA and ramR genes. The acrAB and tolC genes are transcriptionally activated by RamA, the gene for which is itself transcriptionally repressed by RamR. Previous studies have reported that bile induces acrAB in a ramA-dependent manner, but none provided evidence for an induction of ramA expression by bile. Therefore, the objective of this study was to clarify the regulatory mechanism by which bile activates acrAB and tolC. METHODS : qRT-PCR was used to address the effects of bile (using choleate, an ox-bile extract) on the expression of ramA, ramR, acrB and tolC. Electrophoretic mobility shift assays and surface plasmon resonance experiments were used to measure the effect of bile on RamR binding to the ramA promoter (PramA) region. RESULTS : We show that ramA is transcriptionally activated by bile and is strictly required for the bile-mediated activation of acrB and tolC. Additionally, bile is shown to specifically inhibit the binding of RamR to the PramA region, which overlaps the putative divergent ramR promoter, thereby explaining our observation that bile also activates ramR transcription. *
CONCLUSIONS : We propose a regulation model whereby the bile-mediated activation of the acrAB and tolC multidrug efflux genes occurs mainly through the transcriptional derepression of the ramA activator gene.
In Archaea the two major modes of DNA packaging are wrapping by histone proteins or bending by architectural non-histone proteins. To supplement our knowledge about the binding mode of the different DNA-bending proteins observed across the three domains of life, we present here the first model of a complex in which the monomeric Methanogen Chromosomal protein 1 (MC1) from Euryarchaea binds to the concave side of a strongly bent DNA. In laboratory growth conditions MC1 is the most abundant architectural protein present in Methanosarcina thermophila CHTI55. Like most proteins that strongly bend DNA, MC1 is known to bind in the minor groove. Interaction areas for MC1 and DNA were mapped by Nuclear Magnetic Resonance (NMR) data. The polarity of protein binding was determined using paramagnetic probes attached to the DNA. The first structural model of the DNA-MC1 complex we propose here was obtained by two complementary docking approaches and is in good agreement with the experimental data previously provided by electron microscopy and biochemistry. Residues essential to DNA-binding and -bending were highlighted and confirmed by site-directed mutagenesis. It was found that the Arg25 side-chain was essential to neutralize the negative charge of two phosphates that come very close in response to a dramatic curvature of the DNA.
Sedletska, Y. Culard, F. Midoux, P.and Malinge, J. M. (2013)
Interaction studies of muts and mutl with DNA containing the major cisplatin lesion and its mismatched counterpart under equilibrium and nonequilibrium conditionsBiopolymers 99 (9) 636-647
The DNA mismatch repair (MMR) system participates in cis-diamminedichloroplatinum (II) (cisplatin) cytotoxicity through signaling of cisplatin DNA lesions by yet unknown molecular mechanisms. It is thus of great interest to determine whether specialized function of MMR proteins could be associated with cisplatin DNA damage. The major cisplatin 1,2-d(GpG) intrastrand crosslink and compound lesions arising from misincorporation of a mispaired base opposite either platinated guanine of the 1,2-d(GpG) adduct are thought to be critical lesions for MMR signaling. Previously, we have shown that cisplatin compound lesion with a mispaired thymine opposite the 3 platinated guanine triggers new Escherichia coli MutS ATP-dependent biochemical activities distinguishable from those encountered with DNA mismatch consistent with a role of this lesion in MMR-dependent signaling mechanism. In this report, we show that the major cisplatin 1,2-d(GpG) intrastrand crosslink does not confer novel MutS postrecognition biochemical activity as studied by surface plasmon resonance spectroscopy. A fast rate of MutS ATP-dependent dissociation prevents MutL recruitment to the major cisplatin lesion in contrast to cisplatin compound lesion which authorized MutS-dependent recruitment of MutL with a dynamic of ternary complex formation distinguishable from that encountered with DNA mismatch substrate. We conclude that the mode of cisplatin DNA damage recognition by MutS and the nature of MMR post-recognition events are lesion-dependent and suggest that MMR signaling through the major cisplatin lesion is unlikely to occur.
Ovalbumin family contains three proteins with high sequence similarity : ovalbumin, ovalbumin-related protein Y (OVAY), and ovalbumin-related protein X (OVAX). Ovalbumin is the major egg white protein with still undefined function, whereas the biological activity of OVAX and OVAY has not yet been explored. Similar to ovalbumin and OVAY, OVAX belongs to the ovalbumin serine protease inhibitor family (ov-serpin). We show that OVAX is specifically expressed by the magnum tissue, which is responsible for egg white formation. OVAX is also the main heparin-binding protein of egg white. This glycoprotein with a predicted reactive site at Lys367-His368 is not able to inhibit trypsin, plasmin, or cathepsin G with or without heparin as a cofactor. Secondary structure of OVAX is similar to that of ovalbumin, but the three-dimensional model of OVAX reveals the presence of a cluster of exposed positive charges, which potentially explains the affinity of this ov-serpin for heparin, as opposed to ovalbumin. Interestingly, OVAX, unlike ovalbumin, displays antibacterial activities against both Listeria monocytogenes and Salmonella enterica sv. Enteritidis. These properties partly involve heparin-binding site(s) of the molecule as the presence of heparin reverses its anti-Salmonella but not its anti-Listeria potential. Altogether, these results suggest that OVAX and ovalbumin, although highly similar in sequence, have peculiar sequential and/or structural features that are likely to impact their respective biological functions.
Mitogaligin is a mitochondrion-targeting protein involved in cell death. The sequence of the protein is unrelated to that of any known pro- or antiapoptotic protein. Mitochondrial targeting is controlled by an internal sequence from residues 31 to 53, and although this sequence is essential and sufficient to provoke cell death, the precise mechanism of action at the mitochondrial membrane remains to be elucidated. Here, by focusing on the [31–53] fragment, we first assessed and confirmed its cell cytotoxicity by microinjection. Subsequently, with the aid of membrane models, we evaluated the impact of the membrane environment on the 3D structure of the peptide and on how the peptide is embedded and oriented within membranes. The fragment is well organized, even though it does not contain a canonical secondary structure, and adopts an interfacial location. Structural comparison with other membrane-interacting Trp-rich peptides demonstrated similarities with the antimicrobial peptide tritrpcidin.
DNA repair mechanisms are critical for maintaining the integrity of genomic DNA, and their loss is associated with cancer predisposition syndromes. Studies in Saccharomyces cerevisiae have played a central role in elucidating the highly conserved mechanisms that promote eukaryotic genome stability. This review will focus on repair mechanisms that involve excision of a single strand from duplex DNA with the intact, complementary strand serving as a template to fill the resulting gap. These mechanisms are of two general types : those that remove damage from DNA and those that repair errors made during DNA synthesis. The major DNA-damage repair pathways are base excision repair and nucleotide excision repair, which, in the most simple terms, are distinguished by the extent of single-strand DNA removed together with the lesion. Mistakes made by DNA polymerases are corrected by the mismatch repair pathway, which also corrects mismatches generated when single strands of non-identical duplexes are exchanged during homologous recombination. In addition to the true repair pathways, the postreplication repair pathway allows lesions or structural aberrations that block replicative DNA polymerases to be tolerated. There are two bypass mechanisms : an error-free mechanism that involves a switch to an undamaged template for synthesis past the lesion and an error-prone mechanism that utilizes specialized translesion synthesis DNA polymerases to directly synthesize DNA across the lesion. A high level of functional redundancy exists among the pathways that deal with lesions, which minimizes the detrimental effects of endogenous and exogenous DNA damage.
Mismatch-repair factors have a prominent role in surveying eukaryotic DNA-replication fidelity and in ensuring correct meiotic recombination. These functions depend on MutL-homolog heterodimers with Mlh1. In humans, MLH1 mutations underlie half of hereditary nonpolyposis colorectal cancers (HNPCCs). Here we report crystal structures of the MutLα (Mlh1–Pms1 heterodimer) C-terminal domain (CTD) from Saccharomyces cerevisiae, alone and in complex with fragments derived from Mlh1 partners. These structures reveal structural rearrangements and additional domains in MutLα as compared to the bacterial MutL counterparts and show that the strictly conserved C terminus of Mlh1 forms part of the Pms1 endonuclease site. The structures of the ternary complexes between MutLα(CTD) and Exo1 or Ntg2 fragments reveal the binding mode of the MIP-box motif shared by several Mlh1 partners. Finally, the structures provide a rationale for the deleterious impact of MLH1 mutations in HNPCCs.
Matot, B. ; Le Bihan, Y.-V. ; Lescasse, R. ; Pérez, J. ; Miron, S. ; David, G. ; Castaing, B. ; Weber, P. ; Raynal, B. ; Zinn-Justin, S. ; Gasparini, S. and Le Du, M.-H. (2012)
The orientation of the C-terminal domain of the Saccharomyces cerevisiae Rap1 protein is determined by its binding to DNANucleic Acids Res. 40 (7) 3197-3207
Rap1 is an essential DNA-binding factor from the yeast Saccharomyces cerevisiae involved in transcription and telomere maintenance. Its binding to DNA targets Rap1 at particular loci, and may optimize its ability to form functional macromolecular assemblies. It is a modular protein, rich in large potentially unfolded regions, and comprising BRCT, Myb and RCT well-structured domains. Here, we present the architectures of Rap1 and a Rap1/DNA complex, built through a step-by-step integration of small angle X-ray scattering, X-ray crystallography and nuclear magnetic resonance data. Our results reveal Rap1 structural adjustment upon DNA binding that involves a specific orientation of the C-terminal (RCT) domain with regard to the DNA binding domain (DBD). Crystal structure of DBD in complex with a long DNA identifies an essential wrapping loop, which constrains the orientation of the RCT and affects Rap1 affinity to DNA. Based on our structural information, we propose a model for Rap1 assembly at telomere.
Binding of the RamR Repressor to Wild-Type and Mutated Promoters of the ramA Gene Involved in Efflux-Mediated Multidrug Resistance in Salmonella enterica Serovar TyphimuriumAntimicrob Agents Chemother. 56 (2) 942-948
The transcriptional activator RamA is involved in multidrug resistance (MDR) by increasing expression of the AcrAB-TolC RND-type efflux system in several pathogenic Enterobacteriaceae. In Salmonella enterica serovar Typhimurium (S. Typhimurium), ramA expression is negatively regulated at the local level by RamR, a transcriptional repressor of the TetR family. We here studied the DNA-binding activity of the RamR repressor with the ramA promoter (P(ramA)). As determined by high-resolution footprinting, the 28-bp-long RamR binding site covers essential features of P(ramA), including the -10 conserved region, the transcriptional start site of ramA, and two 7-bp inverted repeats. Based on the RamR footprint and on electrophoretic mobility shift assays (EMSAs), we propose that RamR interacts with P(ramA) as a dimer of dimers, in a fashion that is structurally similar to the QacR-DNA binding model. Surface plasmon resonance (SPR) measurements indicated that RamR has a 3-fold-lower affinity (K(D) [equilibrium dissociation constant] = 191 nM) for the 2-bp-deleted P(ramA) of an MDR S. Typhimurium clinical isolate than for the wild-type P(ramA) (K(D) = 66 nM). These results confirm the direct regulatory role of RamR in the repression of ramA transcription and precisely define how an alteration of its binding site can give rise to an MDR phenotype.
Aci-Seche, S., Genest, M. & Garnier, N. (2011)
To address the question of ligand entry process, we report targeted molecular dynamics simulations of the entry of the flexible ionic ligand GW0072 in the ligand binding domain of the nuclear receptor PPARγ. Starting with the ligand outside the receptor the simulations led to a ligand docked inside the binding pocket resulting in a structure very close to the holo-form of the complex. The results showed that entry process is guided by hydrophobic interactions and that entry pathways are very similar to exit pathways. We suggest that TMD method may help in discriminating between ligands generated by in silico docking.
The Drosophila peptidoglycan-recognition protein LF interacts with peptidoglycan-recognition protein LC to downregulate the Imd pathwayEMBO Rep. 12 (4) 327-33 Le Bihan YV, Izquierdo MA, Coste F, Aller P, Culard F, Gehrke TH, Essalhi K, Carell T, Castaing B. (2011)
An alternative flexible conformation of the E. coli HUβ(2) protein : structural, dynamics, and functional aspects.European Biophysics Journal 40 (2) 117-129
The histone-like HU protein is the major nucleoid-associated protein involved in the dynamics and structure of the bacterial chromosome. Under physiological conditions, the three possible dimeric forms of the E. coli HU protein (EcHUα₂, EcHUβ₂, and EcHUαβ) are in thermal equilibrium between two dimeric conformations (N₂ ↔ I₂) varying in their secondary structure content. High-temperature molecular dynamics simulations combined with NMR experiments provide information about structural and dynamics features at the atomic level for the N₂ to I₂ thermal transition of the EcHUβ₂ homodimer. On the basis of these data, a realistic 3D model is proposed for the major I₂ conformation of EcHUβ₂. This model is in agreement with previous experimental data.
Paquet, F., Loth, K., Meudal, H., Culard, F., Genest, D., & Lancelot, G., (2010)
Comparing native and irradiated E. coli lactose repressor-operator complex by molecular dynamics simulation.European Biophysics Journal, 39, 1375-1384.
Fisichella, M., Dabboue, H., Bhattacharyya, S., Saboungi, M.-L., Salvetat J.-P., Hevor, T. and Guérin, M. (2009)
The N-terminal Domain of Drosophila Gram-negative Binding Protein 3 (GNBP3) Defines a Novel Family of Fungal Pattern Recognition Receptors.J. Biol. Chem. 284, 28687-28697. Mishima, Y., Coste, F., Bobezeau, V., Hervouet, N., Kellenberger, C. & Roussel, A. (2009)
Expression, purification, crystallization and preliminary X-ray analysis of the N-terminal domain of GNBP3 from Drosophila melanogaster.Acta Crystallogr. F 65, 870-873. Aci-Seche, S., Garnier, N., Genest, D., Bourg, S., Marot, C., Morin-Allory, L. & Genest, M. (2009)
A Restrained Molecular Dynamics Empirical Approach for Generating a Small Set of Structures Representative of the Internal Flexibility of a Receptor.QSAR Combinatorial Science, 28, 959-968.
Multiple protein structure methods have been proposed for incorporating protein flexibility in molecular docking. One approach for docking ligands onto a rigid receptor is to use an ensemble of multiple rigid structures determined experimentally by X-ray or NMR spectroscopy or generated by numerical simulations. In this work we present all empirical method for generating a wide range of conformational states of a wobbling receptor using restrained Molecular Dynamics simulations (MD) and we propose a partitioning protocol for selecting a few representative conformations of the binding site from restrained MID sampling.
Defining a large number of protein structures is computationally expensive when the MD simulations use an explicit solvent representation. For computational efficiency, solvent effect is therefore represented by an ensemble of restraints applied on a subset of specific atoms, using a distance-dependent permittivity function. The parameters used for the restraints and the permittivity are described. Several 100 ns restrained MD simulations are performed using different sets of parameters. In order to optimize the parameters, the results are compared to a 30 ns MD simulation in explicit solvent. Conformational sampling is speeded up by a factor of around 10-20 when performing restrained MD simulations. A partitioning k-means algorithm is applied to select representative structures of the receptor binding site. The methodology was evaluated on the ligand binding domain of the flexible Peroxysome Proliferator-Activated Receptor-gamma (PPAR gamma).
Genest, D ; Garnier, N ; Arrault, A ; Marot, C ; Morin-Allory, L ; Genest, M (2008)
Ligand-escape pathways from the Ligand Binding Domain of PPAR receptor as probed by molecular dynamics simulationsEuropean Biophysics Journal 37 (4) 369-379
Conformational rearrangements of peroxysome proliferator activated receptor (PPAR ?) ligand-binding domain (LBD) that accompany the release and binding of ligands are not well understood. To determine the major events associated with the escape of the partial agonist GW0072, molecular dynamic (MD) simulations were performed using two different methods : reversed targeted molecular dynamics (TMD-1) and time-dependent distance restraints (TDR) using as restraints either the root mean square deviation from a reference structure (TMD-1) or the distance between the geometrical centers of the binding pocket and of the ligand (TDR). Both methods do not assume any a priori route for ligand extraction.
Insight into the recognition patterns of the ErbB receptor family transmembrane domains : heterodimerization models through MD searchEuropean Biophysics Journal, 37 (6) 851-864.
ErbB receptors undergo a complex interaction network defining hierarchical and competition relationships. Dimerization is driven entirely by receptor-receptor interactions and the transmembrane domains play a role in modulating the specificity and the selection of the partners during signal transduction. To shed light on the role of the GxxxG-like dimerization motifs in the formation of ErbB transmembrane heterodimers, we propose structural models resulting from conformational search method combined with molecular dynamics simulations. Left-handed structures of the transmembrane heterodimers are found preponderant over right-handed structures. All heterotypic heterodimers undergo two modes of association either via the N-terminal motif or the C-terminal motif. The transmembrane domain of ErbB3 impairs this C-terminal motif but also associates with the other partners owing to the presence of Gly residues. The two dimerization modes involve different orientations of the two helices. Thus, a molecular-switch model allowing the transition between the two dimerizing states may apply to the heterodimers and could help interpret receptor competition for the formation of homodimers and heterodimers. The comparison between experimental and theoretical results on the dimerization hierarchy of the transmembrane domains is not straightforward. However, we demonstrate that the intrinsic properties of the transmembrane sequences are an important component in heterodimer formation and that the ErbB2 and ErbB3 transmembrane domains have a strong power for heterodimerization as observed experimentally..
The Methanosarcina thermophila MC1 protein is a small basic protein that is able to bend DNA sharply. When this protein is submitted to oxidative stress through gamma irradiation, it loses its original DNA interaction properties. The protein can still bind DNA but its ability to bend DNA is decreased dramatically. Here, we used different approaches to determine the oxidations that are responsible for this inactivation. Through a combination of proteolysis and mass spectrometry we have identified the three residues that are oxidized preferentially.
Bacterial base excision repair enzyme Fpg (MutM) recognizes bulky N7-substituted-FapydG lesion using a novel and unproductive binding modeChemistry and Biology 15 (7) 706-717 Gutsche, I ; Vujicic-Žagar, A ; Siebert, X ; Servant, P ; Vannier, F ; Castaing, B ; Gallet, B ; Heulin, T ; de Groot, A ; Sommer, S ; Serre, L (2008)
Complex oligomeric structure of a truncated form of DdrA : a protein required for the extreme radiotolerance of DeinococcusBiochimica et Biophysica Acta 1784 (7-8) 1050-1058
PICARD, M., V. BENETEAU, J. BLUM., GUÉRIN, M., CLOIX, J.F., DUFOUR, T., GUILLAUMET, G. and HEVOR, T.K. (2007)
Molecular dynamics simulation approach for the prediction of transmembrane helix-helix heterodimers assembly.European Biophysics Journal, 36, 1071-1082
Computational methods are useful to identify favorable structures of transmembrane (TM) helix oligomers when experimental data are not available or when they cannot help to interpret helix-helix association. We report here a global search method using molecular dynamics (MD) simulations to predict the structures of transmembrane homo and heterodimers. The present approach is based only on sequence information without any experimental data and is first applied to glycophorin A to validate the protocol and to the HER2-HER3 heterodimer receptor.
Picard, M., Guérin, M., Cloix, J.F., Dufour, T. and Hevor, T.K. (2006)
Methionine sulfoximine is well known as a powerful convulsant in many animals. However the actual mechanism of its epileptogenic property is not known. The aim of the present work is to look for a possible effect of methionine sulfoximine on the level of tryptophan because indolamine system was involved in some models of epilepsy. For this Swiss mice were given different doses of methionine sulfoximine and tryptophan concentration was measured using HPLC. During the preconvulsive period no significant change was observed in tryptophan concentration when 100mg/kg of methionine sulfoximine was administered to the animals. Conversely, during the convulsive period, a significant decrease in tryptophan concentration was observed in different regions of the brain including cerebral cortex, striatum, thalamus and cerebellum. During this convulsive period, "grand mal" type seizures were observed. When the animals recovered, no difference was observed in tryptophan concentration between controls and mice submitted to methionine sulfoximine during the post-convulsive period. When different doses of the convulsant were administered the decrease in tryptophan level was significant with 50, 150 and 200mg/kg during the convulsive period, as compared to controls. The present investigation clearly shows a large decrease of about 50% in tryptophan level of mouse brain during the convulsions induced by methionine sulfoximine. Since positron emission tomography reveals also a disturbance in tryptophan utilization in epileptic man, it is possible that tryptophan could be involved in seizure genesis.
Formation of specific complexes between proteins and their cognate DNA modulates the yields and the location of radiation damage on both partners of the complex. The radiolysis of DNA-protein complexes is studied for : (1) the Escherichia coli lactose operator-repressor complex, (2) the complex between DNA bearing an analogue of an abasic site and the repair protein Fpg of Lactococcus lactis. Experimental patterns of DNA damages are presented and compared to predicted damage distribution obtained using an improved version of the stochastic model RADACK. The same method is used for predicting the location of damages on the proteins. At doses lower than a threshold that depends on the system, proteins protect their specific binding site on DNA while at high doses, the studied complexes are disrupted mainly through protein damage. The loss of binding ability is the functional consequence of the amino-acids modification by OH radicals. Many of the most probably damaged amino acids are essential for the DNA-protein interaction and within a complex are protected by DNA.
Transmembrane helix packing of ErbB/Neu receptor in membrane environment : A molecular dynamics studyJournal of Biomolecular Structure & Dynamics 24 (3) 209-228
Dimerization or oligomerization of the ErbB/Neu receptors are necessary but not sufficient for initiation of receptor signaling. The two intracellular domains must be properly oriented for the juxtaposition of the kinase domains allowing trans-phosphorylation. This suggests that the transmembrane (TM) domain acts as a guide for defining the proper orientation of the intracellular domains. Two structural models, with the two helices either in left-handed or in right-handed coiling have been proposed as the TM domain structure of the active receptor.
Bromine-modified oligodeoxynucleotides (ODNs) were fragmented in the electrospray source to study the influence of brominated bases on fragmentation. Several 13-mer ODNs containing a brominated pyrimidine base, BrdU (5-bromodeoxyuridine) or BrdC (5-bromodeoxycytidine), were analyzed. Low cone voltage fragmentation yields a loss of the brominated base with a preferential loss for the brominated base closer to the 5-end (2-position > 4-position > 12-position) as well as a preferential loss of BrdU over BrdC. Higher cone voltage produces backbone fragmentation with complementary a(n)-base and w(m) ions close to the brominated base. On the basis of these observations, we located the brominated base in the sequence for all of the ODNs studied. Copyright (c) 2005 John Wiley & Sons, Ltd.
Begusova, M ; Gillard, N ; Sy, D ; Castaing, B ; Charlier, M ; Spotheim-Maurizot, M (2005)
We discuss here modifications of DNA and protein radiolysis due to the interaction of the-se two partners in specific complexes. Experimental patterns of frank strand breaks (FSB) and alkali revealed breaks (ARB) obtained for DNNA lac operator bound to the lac repressor and for a DNA containing an abasic site analog bound to the formamidopyrimidine-DNA glycosylase are reported. Experimental data are compared to predicted damage distribution obtained using the theoretical model RADACK. (C) 2004 Elsevier Ltd. All rights reserved.
Yhr049w/FSH1 was recently identified in a combined computational and experimental proteomics analysis for the detection of active serine hydrolases in yeast. This analysis suggested that FSH1 might be a serine-type hydrolase belonging to the broad functional alpha beta-hydrolase superfamily. In order to get insight into the molecular function of this gene, it was targeted in our yeast structural genomics project. The crystal structure of the protein confirms that it contains a Ser/His/Asp catalytic triad that is part of a minimal alpha beta-hydrolase fold. The architecture of the putative active site and analogies with other protein structures suggest that FSH1 may be an esterase. This finding was further strengthened by the unexpected presence of a compound covalently bound to the catalytic serine in the active site. Apparently, the enzyme was trapped with a reactive compound during the purification process.
Molecular dynamics (MD) investigations of preformed structures of the transmembrane domain of the oncogenic Neu receptor dimer in a DMPC bilayerBiopolymers 77 (4) 184-197
The critical Val/Glu mutation in the membrane spanning domain of the rat Neu receptor confers the ability for ligand-independent signaling and leads to increased dimerization and transforming ability. There is evidence that the two transmembrane interacting helices play a role in receptor activation by imposing orientation constraints to the intracellular tyrosine kinase domains. By using MD simulations we have attempted to discriminate between correct and improper helix-helix packing by examining the structural and energetic properties of preformed left-handed and right-handed structures in a fully hydrated DMPC bilayer. The best energetic balance between the residues at the helix-helix interface and the residues exposed to the lipids is obtained for helices in symmetrical left-handed interactions packed together via Glu side chain/Ala backbone interhelical hydrogen bonds.
The MC1 protein is a chromosomal protein likely involved in the DNA compaction of some methanogenic archaea. This small and monomeric protein, structurally unrelated to other DNA binding proteins, bends DNA sharply. By studying the protein binding to various kinds of kinked DNA, we have previously shown that MC1 is able to discriminate between different deformations of the DNA helix. Here we investigate its capacity to recognize particular DNA sequences by using a SELEX procedure. We find that MC1 is able to preferentially bind to a 15 base pair motif [AAAAACACAC(A/C)CCCC]. The structural parameters of this sequence are characterized by molecular dynamics simulation experiments, and the binding mode of the protein to the DNA is studied by footprinting experiments. Our results strongly suggest that the protein realizes an indirect readout of the DNA sequence by binding to the DNA minor groove.
The chromosomal protein MC1 is a monomeric protein of 93 amino acids that is able to bind any DNA but has a slight preferential affinity for some sequences and structures, like cruciform and minicircles. The protein has been irradiated with Ar-36(18+) ions of 95 MeV/nucleon. The LET of these particles in water is close to 270 keV/mu m. We tested the activity of the protein by measuring its ability to form complexes with DNA. We tested the integrity of the protein by measuring the molecular weight of the species formed. Compared with gamma radiation, we observed for the same dose a less efficient inactivation of the protein, a greater protection of the protein by the bound DNA, a lower induction of chain breakage, and a greater production of protein-protein and DNA-protein crosslinks. The results are discussed in terms of the quantitative and the qualitative differences between the two types of radiation : The global radical yield is slightly higher with gamma rays, whereas the density of radicals produced along the particle track is considerably higher with argon ions. (c) 2005 by Radiation Research Society.
Structural insights into abasic site for Fpg specific binding and catalysis : comparative high-resolution crystallographic studies of Fpg bound to various models of abasic site analogues-containing DNANucleic Acids Research 33 (18) 5936-5944
dFpg is a DNA glycosylase that recognizes and excises the mutagenic 8-oxoguanine (8-oxoG) and the potentially lethal formamidopyrimidic residues (Fapy). Fpg is also associated with an AP lyase activity which successively cleaves the abasic (AP) site at the 3’ and 5’ sides by ß delta-elimination. Here, we present the high-resolution crystal structures of the wild-type and the P1G defective mutant of Fpg from Lactococcus lactis bound to 14mer DNA duplexes containing either a tetrahydrofuran (THF) or 1,3-propanediol (Pr) AP site analogues.
Gillard, N ; Begusova, M ; Castaing, B ; Spotheim-Maurizot, M (2004)
During the base excision repair of certain DNA lesions, the formamidopyrimidine-DNA glycosylase (Fpg) binds specifically to the DNA region containing an abasic (AP) site. Is this step affected by exposure to ionizing radiation ? To answer this question, we studied a complex between a DNA duplex containing an analogue of an abasic site (the 1,3-propanediol site, Pr) and a mutated Lactococcus lactis Fpg (PIG-LlFpg) lacking strand cleavage activity. Upon irradiation of the complex, the ratio of bound/free partners decreased. When the partners were irradiated separately, the irradiated DNA still bound the unirradiated protein, whereas irradiated Fpg no longer bound unirradiated DNA.
The three-dimensional structure of methanogen chromosomal protein 1 (MC 1), a chromosomal protein extracted from the archaebacterium Methanosarcina sp. CHT155, has been solved using H-1 NMR spectroscopy. The small basic protein MC1 contains 93 amino acids (24 basic residues against 12 acidic residues). The main elements of secondary structures are an a helix and five ß strands, arranged as two antiparallel ß sheets (a double one and a triple one) packed in an orthogonal manner forming a barrel. The protein displays a largely hydrophilic surface and a very compact hydrophobic core made up by side chains at the interface of the two ß sheets and the helix side facing the interior of the protein.
Structural basis for the recognition of the FapydG lesion (2,6-diamino-4-hydroxy-5-formamidopyrimidine) by formamidopyrimidine-DNA glycosylaseJournal of Biological Chemistry 279 (42) 44074-44083
Formamidopyrimidine-DNA glycosylase (Fpg) is a DNA repair enzyme that excises oxidized purines such as 7,8-dihydro-8-oxoguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) from damaged DNA. Here, we report the crystal structure of the Fpg protein from Lactococcus lactis (LlFpg) bound to a carbocyclic FapydG (cFapydG)-containing DNA.
Insights into the DNA repair process by the formamidopyrimidine-DNA glycosylase investigated by molecular dynamicsProtein Science 13 (8) 2009-2021
Formamidopyrimidine-DNA glycosylase (Fpg) identifies and removes 8-oxoguanine from DNA. All of the X-ray structures of Fpg complexed to an abasic site containing DNA exhibit a common disordered region present in the C-terminal domain of the enzyme. However, this region is believed to be involved in the damaged base binding site when the initial protein/DNA complex is formed. The dynamic behavior of the disordered polypeptide (named Loop) in relation to the supposed scenario for the DNA repair mechanism was investigated by molecular dynamics on different models, derived from the X-ray structure of Lactococcus lactis Fpg bound to an abasic site analog-containing DNA and of Bacillus stearothermophilus Fpg bound to 8-oxoG.
Garnier, N., Crouzy, S. & Genest, M. (2003)
Molecular Dynamics Simulations of the Transmembrane Domain of the Oncogenic ErbB2 Receptor Dimer in a DMPC Bilayer.Journal of Biomolecular Structure and Dynamics 21, 179-199. Déclais, A. C., Fogg, J. M., Freeman, A. D. J., Coste, F., Hadden, J. M., Phillips, S. E. V. & Lilley, D. M. J. (2003)
The DNA-binding protein MC1 is a chromosomal protein extracted from the archaebacterium. Methanosarcina sp. CHT155. It binds any DNA, and exhibits an enhanced affinity for some short sequences and structures (circles, cruciform DNA). Moreover, the protein bends DNA strongly at the binding site. MC1 was submitted to oxidative stress through gamma-ray irradiation. In our experimental conditions, damage is essentially due to hydroxyl radicals issued from water radiolysis. Upon irradiation, the regular complex between MCl and DNA disappears, while a new complex appears. In the new complex, the protein loses its ability to recognise preferential sequences and DNA circles, and bends DNA less strongly than in the regular one. The new complex disappears and the protein becomes totally inactivated by high doses.
Several purine and pyrimidine cyclonucleosides were found to be not recognized by several Escherichia coli and yeast DNA N-glycosylases. Interestingly, a non covalent complex was observed between the Lactoccocus lactis formamido-pyrimidine-DNA glycosylases (Fpg-L1) and the cyclonucleosides. This may provide new information on the mechanism involved in the activity of the latter enzyme.
Evidence of a thermal unfolding dimeric intermediate for the Escherichia coli histone-like HU proteins : Thermodynamics and structureJournal of Molecular Biology 331 (1) 101-121
The Escherichia coli histone-like HU protein pool is composed of three dimeric forms : two homodimers, EcHUa(2) and EcHUß(2), and a heterodimer, EcHUaß. The relative abundance of these dimeric forms varies during cell growth and in response to environmental changes, suggesting that each dimer plays different physiological roles.
Charlier, M ; Eon, S ; Seche, E ; Bouffard, S ; Culard, F ; Spotheim-Maurizot, M (2002)
Upon gamma-ray or argon ion irradiation of the lac repressor protein, its peptide chain is cleaved and the protein loses its lac operator-binding activity, as shown respectively by polyacrylamide gel electrophoresis and retardation gel ejectrophoresis. We developed phenomenological models that satisfactorily account for the experimental results : the peptide chain cleavage model considers that the average number of chain breaks per protomer is proportional to the irradiation dose and that the distribution of the number of breaks per protomer obeys Poisson’s law.
Crystallization and preliminary X-ray crystallographic studies of a complex between the Lactococcus lactis Fpg DNA-repair enzyme and an abasic site containing DNAActa Crystallographica Section D-Biological Crystallography 58 679-682 Part 4
For protein-DNA complex crystallization, the choice of the DNA fragment is crucial. With the aim of crystallizing the 31 kDa Fpg DNA-repair enzyme bound to DNA, oligonucleotide duplexes varying in length, sequence, end type and nature of the specific DNA target site were used. Crystals of several protein-DNA combinations grew from solutions containing both polyethylene glycol and salt. This systematic crystallization screening followed by optimization of the crystallization conditions by microseeding led to crystals of Fpg bound to a 13 base-pair duplex DNA carrying the 1,3-propanediol abasic site analogue which are suitable for crystallographic analysis. Complete native data sets have been collected to 2.1 Angstrom resolution.
Description of ordered solvent molecules in a platinated decanucleotide duplex refined at 1.6A resolution against experimental MAD phases.Acta Crystallographica Section D-Biological Crystallography 58 431-440
Accurate experimental phases derived from a MAD experiment may be useful to enable the identification of solvent molecules during the course of an atomic parameter refinement. The structure of a double-stranded DNA decanucleotide bearing a cisplatin interstrand cross-link at 1.6 Angstrom resolution, whose phases were first determined experimentally using the L-III edge of the Pt atom, was refined by various methods. The previously published structure resulted from a least-squares refinement using the structure-factor magnitudes and stereochemical restraints (program SHELX). In this paper, these previous results are compared with a model obtained by the likelihood-maximization method (program REFMAC) which allows the combination of the observed magnitudes with experimental MAD phases.
Crystal structure of the Lactococcus lactis formamidopyrimidine-DNA glycosylase bound to an abasic site analogue-containing DNAEmbo Journal 21 (12) 2854-2865
The formamidopyrimidine-DNA glycosylase (Fpg, MutM) is a bifunctional base excision repair enzyme (DNA glycosylase/AP lyase) that removes a wide range of oxidized purines, such as 8-oxoguanine and imidazole ring-opened purines, from oxidatively damaged DNA. The structure of a non-covalent complex between the Lactoccocus lactis Fpg and a 1,3-propanediol (Pr) abasic site analogue-containing DNA has been solved. Through an asymmetric interaction along the damaged strand and the intercalation of the triad (M75/R109/F111), Fpg pushes out the Pr site from the DNA double helix, recognizing the cytosine opposite the lesion and inducing a 60degrees bend of the DNA.
Begusove, M ; Eon, S ; Sy, D ; Culard, F ; Charlier, M ; Spotheim-Maurizot, M (2001)
Purpose : To calculate the probability of radiation-induced frank strand breakage (FSB) at each nucleotide in the Escherichia coli lac repressor-lac operator system using a simulation procedure. To compare calculated and experimental results. To asses the contribution of DNA conformational changes and of the masking by the protein to DNA protection by the repressor. Materials and methods : Two structures of the complex were extracted from the PDB databank : crystallography- and NMR-based structures. Calculations were made of the accessibility of the atoms mainly involved in strand breakage (H4’ and H5’) to OH* and of the FSB probabilities, along : (1) DNA in the complex ; (2) DNA in the complex depleted of the repressor ; and (3) a linear DNA having the same sequence. An 80 bp fragment bearing the operator was irradiated alone or in presence of the repressor.
Radiation disrupts protein-DNA complexes through damage to the protein. The lac repressor-operator systemRadiation Research 156 (1) 110-117
Binding of a protein to its cognate DNA sequence is a key step in the regulation of gene expression. If radiation damage interferes with protein-DNA recognition, the entice regulation profess may be perturbed. We have studied the effect of gamma rays on a model regulatory system, the E. coli lactose repressor-operator complex. We have observed the disruption of the complex upon irradiation in aerated solution. The complex is completely restored by the addition of nonirradiated repressor, hut not by the addition of nonirradiated DNA.
Le Cam, E ; Delain, E ; Larquet, E ; Culard, F ; Cognet, JAH (2000)
Catalytic and DNA binding properties of the Ogg1 protein of Saccharomyces cerevisiae : Comparison between the wild type and the K241R and K241Q active-site mutant proteinsBiochemistry 39 (7) 1716-1724
The Ogg1 protein of Saccharomyces cerevisiae belongs to a family of DNA glycosylases and apurinic/apyrimidinic site (AP) lyases, the signature of which is the a-helix-hairpin-a-helix-Gly/Pro-Asp (HhH-GPD) active site motif together with a conserved catalytic lysine residue, to which we refer as the HhH-GPD/K family. In the yeast Ogg1 protein, yOgg1, the HhH-GPD/K motif spans residues 225-260 and the conserved lysine is K241. In this study, we have purified the K241R and K241Q mutant proteins and compared their catalytic and DNA binding properties to that of the wild-type yOgg1. The results show that the K241R mutation greatly impairs both the DNA glycosylase and the AP lyase activities of yOgg1.