Generation of the primitive neuroectoderm into specialized brain subdivisions, such as the hindbrain primordium, involves the regulated coordination of complex morphogenetic and molecular mechanisms. These processes are evident in the segregation of the zebrafish hindbrain into seven distinct lineage-restricted compartments, termed rhombomeres (r), which are established by the interplay of several spatially-restricted expressed genes. These include transcription factors, members of specific signaling pathways and specialized molecules that mediate cell adhesion and identity. Despite their extensive characterization, it is evident that other genes are involved to mediate the proper specification and segregation of individual rhombomeres. One candidate that likely fits this role is related to the no ocelli/l(2)35Ba gene in Drosophila, termed nlz (nocA-like zinc-finger). Nlz-related proteins behave as transcriptional repressors and are related to the vertebrate Sp1-like family of transcription factors. nlz is dynamically expressed in the zebrafish hindbrain, residing in the caudal hindbrain at gastrula stages and rostrally expanding from presumptive r3/r4 boundary to encompass r3 and r2 at segmentation stages. Nlz localizes to the nucleus and associates with the co-repressors Groucho and histone deacetylases, suggesting that Nlz acts as a repressor. Consistent with this, misexpression of nlz into zebrafish embryos results in a loss of gene expression in the rostral hindbrain (rl-r3). Taken together, the findings in this thesis suggest that Nlz functions as a transcriptional repressor to control segmental gene expression in the rostral hindbrain.

Fur (Ferric Uptake Regulator) est un régulateur transcriptionnel spécifique des bactéries qui intervient dans le contrôle de l'homéostasie du fer, ce qui en fait une cible antibactérienne intéressante. Avant mon arrivée au laboratoire, quatre inhibiteurs interagissant spécifiquement avec Fur avaient été isolés. La partie active de ces inhibiteurs consiste en des peptides de 13 acides aminés. Au cours de cette thèse, j'ai utilisé une double-approche : théorique et expérimentale pour étudier l'interaction de ces peptides avec Fur afin de comprendre le mécanisme d'inhibition. J'ai synthétisé plusieurs séquences peptidiques, montré par des tests biochimiques que certaines inhibaient Fur et déterminé les interactions importantes à l'activité inhibitrice. J'ai obtenu des modèles théoriques des complexes Fur/peptides par amarrage moléculaire, cohérents avec les résultats expérimentaux, qui ont mis en évidence une zone d'inhibition de Fur. Des criblages in silico dans cette zone ont permis de sélectionner de petites molécules, inhibitrices potentielles de Fur et donc intéressantes pour des applications thérapeutiques. / Fur (Ferric Uptake Regulator) is a transcriptional regulator involved in the control of iron homeostasis. Specific to bacteria, Fur is an attractive antibacterial target. Before my arrival in the laboratory, four inhibitors interacting specifically with Fur had been isolated. The active part of these inhibitors consists of peptides of 13 amino acids. In this thesis I have used both theoretical and experimental approaches to study interactions of these peptides with Fur in order to understand the inhibition mechanism. I have synthesized several peptide sequences, shown through biochemical assays that some of them could inhibit Fur and I have identified residues important to the inhibitory activity. I‘ve obtained theoretical models of Fur/peptide complexes consistent with experimental results, which reveal an inhibition pocket in Fur. Small molecules have then been selected though In silico screening of this pocket, that could potentially inhibit Fur, and thus be interesting for therapeutic applications.

The present thesis deals with different aspects of the chemistry and photo-biology of various ferrocene-conjugates, their interaction with double helical DNA, DNA photocleavage and photo-enhanced cytotoxicity in visible light, localization and cellular uptake to study the mechanism of cell death. Phenyl analogues of the active complexes have been synthesized and used for comparison in biological assays. Chapter I presents an overview of cancer and its types, various treatments for cancer. A general overview on the Photodynamic Therapy, a new modality of light activated cancer treatment and its various possible mechanism of action, has been made. The promise of photoactivated chemotherapy is discussed with recently developed metal based antitumor agents. Biological applications of few ferrocene conjugates as anticancer and anti-malarial agents are discussed. The objective of the present investigation is also presented in this chapter. Chapter II presents the synthesis, characterization, structure, DNA binding, DNA photocleavage, photocytotoxicity and cellular localization of ferrocene-conjugated dipicolylamine oxovanadium(IV) complexes of curcumin. To explore the role of the ferrocenyl moiety the phenyl analogue of the ferrocenyl complexes is synthesized and used as a control for comparison purpose. Chapter III deals with the photo-induced DNA cleavage and photo-enhanced cytotoxicity of ferrocene-conjugated oxovanadium(IV) complexes of heterocyclic bases. The synthesis, characterization, structural comparisons, DNA binding, DNA photocleavage and photocytotoxic activity in visible light are discussed in detail. Chapter IV describes the synthesis, characterization and structure of ferrocene-conjugated oxovanadium(IV) complexes of acetylacetonate derivatives. The complexes are evaluated for DNA binding, DNA photocleavage and photocytotoxic activity in HeLa, MCF-7, 3T3 cells in visible light. The fluorescent nature of the complexes is used to study the cellular localization of the complexes and the mechanism of cell death induced by the complexes is also discussed. Chapter V presents the photocytotoxic effect of ferrocene-conjugated oxovanadium(IV) complexes of different curcuminoids in HeLa , HepG2 and 3T3 cells. Curcumin based fluorescence has been successfully used to study the cellular uptake and localization behavior of the complexes. The positive role of the ferrocenyl complex is evident from the ~4 fold increase in its photocytotoxicity compared to the phenyl analogue. The apoptotic mode of cell death is evident from nuclear co-staining using Hoechst dye. Chapter VI describes the synthesis, characterization and photochemotherapeutic efficacy of ferrocene conjugates of N-alkyl pyridinium salts. Mitochondria targeting property of ferrocene compound having n-butyltriphenylphosphonium group has been studied by JC-1 assay. FACS analysis showed significant sub G1/G0 phase cell-cycle arrest in cancer cells on visible light treatment. Finally, the summary of the dissertation and conclusions drawn from the present investigations are presented. The references in the text have been indicated as superscript numbers and compiled at the end of each chapter. The complexes presented in this thesis are represented by bold-faced numbers. Crystallographic data of the structurally characterized complexes are given in CIF format in the enclosed CD (Appendix-I). Due acknowledgements have been made wherever the work described is based on the findings of other investigators. Any unintentional omission that might have happened due to oversight or mistake is regretted. INDEX WORDS: Ferrocene conjugates Crystal structure DNA binding DNA photocleavage Photocytotoxicity Vanadium Cellular Imaging

Photodynamic therapy(PDT) has recently emerged as a promising new non-invasive treatment modality for a large number of neoplastic and non-neoplastic lesions. Photoexcitation of a photosensitizing drug in the tumor tissue causes generation of reactive oxygen species which results in cell death. The current porphyrinic photosensitizers suffer a wide range of drawbacks leading to the development of the chemistry of alternative photosensitizing agents in PDT. Among them, the 4d and 5d transition metal-based photosensitizers have been explored extensively with the exception of the 3d metal complexes. The objective of this thesis work is to design and synthesize photoactive iron(III) abd cobalt(III) complexes and evalutate their photonuclease and photocytotoxic potential. Bioessential 3d metal ions provide an excellent platform for metal-based PDT drug designing as because of its varied spectral, magnetic and redox properties, with its complexes possessing rich photochemical behavior in aqueous and non-aqueous media. We have synthesized binary iron(III) complexes as netropsin mimics using amino acid Schiff bases derived from salicylaldehyde/napthaldehyde and arginine/lysine. The complexes were found to be good AT selective DNA binders and exhibited significant DNA photocleavage activity. To enhance the photodynamic potential, we further synthesized iron(III) complexes of phenolate-based ligand and planar phenanthroline bases. The DNA photocleavage activity of these complexes and their photocytotoxic potential in cancer models were studied. ROS generated by these complexes were found to induce apoptotic cell death. Ternary cobalt(III) complexes were synthesized to study the effect of the central metal atom. The diamagnetic cobalt(III) complexes were structurally dissimilar to their iron(III) analogues. Although the Co(III)/Co(II) redox couple is chemically and photochemically accessible but the Co(III)-dppz complex, unlike its iron(III)-dppz analogue, exhibited selective damage to hTSHR expressing cells but not in HeLa cells. A structure-activity relationship study on iron(III) phenolates having modified dppz ligands was carried out and it was found that electron donating group on the phenazine unit and an increase of the aromatic surface area largely improved the PDT efficiency. Finally, SMVT targeted iron(III) complexes with biotin as targeting moiety were synthesized and the in vitro efficacy of the complexes was tested in HepG2 cells over-expressing SMVTs and compared to HeLa amd HEK293 cells. The complexes exhibited higher phytocytotoxicity in HepG2 than in HeLa and cells and HEK293 cells. An endocytotic mode of uptake took place in HepG2 cells whereas in HEK293 cells, uptake is purely by diffusion. This is expected to reduce the side-effects and have less effect on cells with relatively less SMVTs. In summary, the present research work opens up novel strategies for the design and development of primarily iron-based photosensitizers for their potential applications in PDT with various targeting moieties.

DNA repair proteins in mycobacteria and their physiological importance Mycobacterium tuberculosis, the causative organism of tuberculosis, resides in the host macrophages where it is subjected to a plethora of stresses like reactive oxygen species (ROS) and reactive nitrogen intermediate(RNI) which are generated as a part of the host’s primary immune response. These stresses can damage the cellular components of the pathogen including DNA and its precursors. Two common damages to DNA and its precursors caused by ROS and RNI are oxidation of guanine to 8-oxo-guanine and deamination of cytosine to uracil. Mycobacteria, which are known to have high G+C content, must be more susceptible to such damages, and are thus equipped with the mechanisms to counteract these damages. One such mechanism is to hydrolyse the 8-oxo-dGTP into 8-oxo-dGMP to avoid its incorporation in the DNA during its synthesis. This job is done by a protein called MutT.In mycobacteria four homologs of MutT, namely MutT1, MutT2, MutT3 and MutT4 have been annotated. The second mechanism deals with the repair of uracil residues present in DNA which are generated by deamination of cytosines or incorporation of dUTP during DNA synthesis. This is taken care of by a protein called uracil DNA glycosylase (UDG) which excises uracil by cleaving the N-C1’ glycosidic bond between the uracil and the deoxyribose sugar in a DNA repair pathway called the base excision repair (BER). In this study, the biochemical properties and physiological role of mycobacterial MutT2 and, MSMEG_0265 (MsmUdgX), a novel uracil DNA glycosylase superfamily protein, have been investigated. I.Biochemical characterization of MutT2 from mycobacteria and its antimutator role. Nucleotide pool, the substrate for DNA synthesis is one of the targets of ROS which is generated in the macrophage upon Mycobacterium tuberculosis infection. Thus, the pathogen is at increased risk of accumulating oxidised guanine nucleotides such as 8-oxo-dGTP and 8-oxo-GTP. By hydrolysing the damaged guanine nucleotides before their incorporation into nucleic acids, MutT proteins play a critical role inallowing organisms to avoid their deleterious effects. Mycobacteria possess several MutT proteins. Here, we have purified recombinantM. tuberculosisMutT2 (MtuMutT2) andM. smegmatisMutT2 (MsmMutT2) proteins as representative of slow and fast growing mycobacteria, for the purpose of biochemical characterization. UnlikeEscherichia coliMutT, which hydrolyzes 8-oxo-dGTP and 8-oxo-GTP, the mycobacterial proteins hydrolyze not only 8-oxo-dGTP and 8-oxo-GTP but also dCTP and 5-methyl-dCTP. Determination of kinetic parameters (KmandVmax) revealed thatwhileMtuMutT2 hydrolyzes dCTP nearly four times better than it does 8-oxo-dGTP,MsmMutT2 hydrolyzes them almost equally well. Also,MsmMutT2 is about 14 times more efficient thanMtuMutT2 in its catalytic activity of hydrolyzing 8-oxo-dGTP.Consistent with these observations,MsmMutT2 but notMtuMutT2 rescuesE. colifor MutT deficiency by decreasing both themutation frequency and A to C mutations (a hallmark of MutT deficiency). We discuss these findings in the context of the physiological significance of MutT proteins. II.Understanding the biochemical properties of MSMEG_0265 (MsmUdgX), a novel uracil DNA glycosylase superfamily protein Uracil DNA glycosylases (UDGs) are base excision repair enzymes which excise uracil from DNA by cleaving the N-glycosidic bond. UDGs are classified into 6 different families based on their two functional motifs, i. e.,motif A and motif B. In mycobacteria, there are two uracil DNA glycosylases, Ung and UdgB which belong to Family 1 and Family 5, respectively. In this study, based on the presence of the two functional motifs, we have discovered yet another uracil DNA glycosylase in M. smegmatis, which we have called MsmUdgX.The motif A and motif B of this protein indicate that it does not belong to any of the UDG families already classified but has highest similarity with Family 4 UDGs. Homologs of this protein are also present in several other organisms like M. avium, Streptomyces ceolicolor, Rhodococcus etc., but absent in M. tuberculosis, archaea and eukaryotes. Activity assays of this protein show that unlike other UDGs, MsmUdgX does not excise uracil, but forms a tight complex with uracil containing single stranded (ss) and double stranded (ds) DNAs, as observed by a shifted band in 8M urea-PAGE as well as SDS-PAGE. It also does not recognize other modified nucleotides that we investigated, in DNA. The protein binds to uracil-DNA in a wide range of pH and the minimum substrate required for its binding is pNUNN. Like Family 4 UDG, the protein has Fe-S cluster but it is not as thermostable as the Family 4 UDGs. Addition of different metal ions does not affect its binding property, and even the presence of M. smegmatis cell free extract does not diminish its binding activity. Since this protein binds specifically to uracil in DNA, an application of the protein for detection of uracil in the genomic DNA is proposed. III. Elucidation of the role of KRRIH loop in MsmUdgX by mutational analysis MsmUdgX is a novel uracil DNA glycosylase superfamily protein which has the highest homology to Family 4 UDGs. However, alignment of MsmUdgX amino acid sequence with that of Family 4 UDGs shows that there is an extra stretch of amino acids which is unique to this group of proteins. This stretch, defined by AGGKRRIH is absent in all Family 4 UDGs and the region KRRIH of the strtch is quite conserved amongst all UdgX proteins. Homology modelling of MsmUdgX, using a Family 4 UDG (TthUdgA) shows that this extra stretch of amino acids forms an outloop near the enzyme active site. Another unique difference between MsmUdgX and Family 4 UDGs is in the motif A where MsmUdgX has GEQPG and the Family 4 UDGs haveGE(A/G)PG. Our work on MsmUdgX has shown that, unlike other UDGs, this protein does not excise uracils, but forms a tight complex with the uracil containing DNA. This unique tight uracil binding property as well as KRRIH amino acid stretch has not been observed for any uracil DNA glycosylase superfamily proteins. So, to gain insight into the role of KRRIH and glutamine (Q) of motif A in MsmUdgX family of proteins, site directed mutagenesis was done in this region and we observed that mutation of His109 of the KRRIH loop to serine (S) leads to a gain of uracil excision activity, whereas changing the R107 to S, ‘RRIH’ to ‘SSAS’ or deleting the loop altogether leads to loss of its complex formation activity. Further, mutation of H109 to other amino acids like G, Q and A also shows uracil excision activity. Mutation of the glutamine in the motif A to alanine so that it is exactly similar to that of Family 4 UDGs, does not affect its uracil binding activity. This observation indicates that the KRRIH loop has an important role in the tight binding and/or uracil excision activity of MsmUdgX. Crystal structure of MsmUdgX in complex with uracil-DNA oligo and MsmUdgX H109S mutants are being studied.IV. Physiological importance of MsmUdgX in M. smegmatis MsmUdgX is a uracil DNA glycosylase superfamily protein which binds tightly to uracil (in DNA) without excising it. To elucidate its role in M. smegmatis, knockout of udgX was generated. Growth comparison of the wild type and the ΔudgX strains does not show any growth differences under the conditions tested. However, overexpression of MsmUdgX in recA deficient strains of E. coli as well as M. smegmatis leads to their retarded growth. Retarded grown is also observed in strains deficient in other DNA repair proteins that work in conjunction with RecA. These observations indicate that repair/release of MsmUdgX-uracil DNA complex might be a RecA dependent process.

Structural Maintenance of Chromosomes (SMC) proteins are a highly conserved class of proteins required for the maintenance of genome stability and regulate nearly all aspects of chromosome biology. Eukaryotes, such as the budding yeast Saccharomyces cerevisiae, have six Smc proteins that form three SMC complexes in association with non-SMC proteins, i.e., the cohesin complex, the condensin complex and the Smc5/6 complex. The yeast Smc5/6 complex consists of Smc5, Smc6 and six non-Smc elements (Nse1-6) that are all essential for the survival of cells. Nse1 is the first non-smcelement that was identified associated with the Smc5/6 complex. Nse1 has a C-terminal RING-domain, which is a characteristic feature of some E3 ubiquitin ligases. A RING domain consists of eight conserved Zn-coordinating residues arranged in a cross-brace conformation. To understand the importance of this domain, we created site directed mutations in conserved residues identified by sequence alignment of the budding yeast Nse1 RING domain with that of other species. We found a new RING domain mutant nse1-103that was temperature sensitive at 37°C and showed an increased sensitivity towards genotoxic agents such as hydroxyurea (HU), methyl methane sulfonate (MMS) and ultraviolet (UV) radiation. Thense1-103 mutant cells are slow growing and show delayed chromosomal replication at the restrictive temperature. Genetic interactions with replication factors such as RRM3, TOF1 etc. revealed thatnse1-103shows a synthetic sick growth defect in combination with rrm3∆ that is partially suppressed by deletion of TOF1. We found an enhancement in chromosome loss in nse1-103 compared to wild type cells. This was accompanied by a slight reduction in cohesion between the sister chromatids in nse1-103,suggesting a plausible mechanism for the chromosome destabilization observed in the mutant. Since Nse1 forms part of a trimeric sub-complex with Nse3 and Nse4 in the Smc5/6 complex, we performed a yeast two hybrid assay to test the interaction of nse1-103 with Nse3 or Nse4, and found a defect in interaction of nse1-103 with Nse3 and Nse4. In addition, a defect in association of nse1-103 with Smc5 or Smc6 could be observed by performing co-immunoprecipitation from yeast cell lysates, suggesting that the integrity of the RING-domain is critical for the interaction of Nse1 with other subunits of the Smc5/6 complex. However, there was no defect in the interaction between Nse3 and Smc5 in nse1-103, indicating that the interaction of these components within the complex isindependent of Nse1. We also identified a novel sequence motif near the RING domain of Nse1, deletion of which leads to an increased sensitivity towards genotoxic stressors and higher temperature. Biochemical characterization of this mutant also suggests a defect ininteraction with Nse3 or Nse4, and also with Smc5. The nse1 mutants also showed defects in post translational modification of Smc5 and other proteins. Since the Smc5/6 complex also has a SUMO E3 ligase, Mms21/Nse2, we also investigated genetic interactions between the RING domain mutant,nse1-103 and the SUMO ligase RING domain defective mutant,mms21∆sl, and found an exacerbation of the drug sensitive phenotypes in thense1-103 mms21∆sl double mutant relative to either of the single mutants nse1-103 or mms21∆sl, indicating that the two proteins contribute independently to the function of Smc5/6 complex in resisting genotoxic stress. In conclusion, the present study emphasizes the role of the RING domain of budding yeast Nse1 in resisting genotoxic stress and maintaining chromosome stability and reveals that the integrity of the RING-domain is critical for interactions of Nse1 with Nse3 and other Smc5/6 complex components. In addition, we report identification of another novel sequence motif in Nse1 that is also crucial for its interaction with other subunits of the Smc5/6 complex and for maintenance of post-translational modifications of some cellular proteins.

Les facteurs de transcription de la famille Rel/NF-κB régulent l’expression d’un grand nombre de gènes impliqués dans les réponses immunitaires et inflammatoires ainsi que dans la régulation de la prolifération et de la survie cellulaire. Le caractère transitoire de l’activation de NF-κB est donc crucial pour poterger les cellules de l’autoxicité due à une trop forte expression des gènes cibles de ce facteur de transcription. Dans le cadre de notre thèse de doctorat, nous avons étudié les mécanismes moléculaires régulant la cinétique d’activation de NF-κB, en accordant une attention toute particulière au complexe kinase IKK, qui semble être le regulateur clef de l’activation de NF-κB. Nos résultats suggèrent que p300 pourrait réguler la durée d’activation des IKKs d’une part par acétylation directe, et d’autre part, indépendamment de son activité HAT, en stabilisant les IKKs et donc en prolongeant leur demie-vie et par conséquent leur activation.<p>Certains virus utilisent la voie de signalisation NF-κB afin de promouvoir leur propre réplication. C’est le cas du virus HIV-1 (Human Immunodeficiency Virus type 1), qui contient dans son promoteur deux sites de liaison pour NF-κB. Notre laboratoire a précédemment montré que l’utilisation du TNFα en combinaison avec la TSA, active l’expression virale de manière synergique. L’administration combinée d’un activateur du facteur NF-κB et d’un inhibiteur de désacétylases pourrait, en présence d’une thérapie anti-HIV-1 efficace, être envisagée dans le but d’éliminer les cellules réservoirs infectées de manière latente. L’utilisation thérapeutique du TNFα ou de la TSA étant inenvisageable en raison de leur toxicité, nous avons étudié l’effet d’autres substances ayant un plus grand potentiel thérapeutique et nous avons apporté une preuve de principe du potentiel thérapeutique de la coadministration de plusieurs activateurs viraux (inhibiteurs de HDACs[HDACIs]+inducteurs de la voie NF-κB) pour réduire le pool des réservoirs cellulaires infectés de manière latente.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

La réactivation aberrante du gène TWIST1 embryonnaire a été identifiée dans le laboratoire comme un mécanisme d'inactivation récurrente des voies dépendantes de p53 et Rb dans de nombreuses tumeurs. En diminuant la sénescence et l’induction de l'apoptose, TWIST1 coopère avec des protéines oncogéniques dans la transformation cellulaire in vitro et intervient dans l'initiation et la progression tumorale in vivo. Comme TWIST1 est très faiblement exprimée dans la plupart des cellules adultes différenciées, elle constitue une cible attrayante pour des thérapeutiques futures. Récemment, l’héterodimère TWIST1/E2A (E12 ou E47, deux produits d'épissage alternatif du gène TCF3) joue un rôle pro-métastatique dans le cancer de la prostate (Gajula et al., 2015), alors que ce complexe est la forme oncogénique de TWIST1 dans des cellules épithéliales mammaires humaines (Jacqueroud et al., 2016). L'hétérodimérisation par l'intermédiaire des domaines HLH est une condition sine qua non pour la formation de domaine de liaison d'ADN (Murre et Massari 2000). Une approche in silico basé que la modélisation par homologie des complexes de TWIST1 met en évidence le rôle déterminant des boucles inter-hélicales dans le maintien de complexes de TWIST1 à l’ADN en étudiant plusieurs insertions de TWIST1 observées chez les patients atteints de syndrome Saethre-Chotzen (Bouard et al., 2014). Ensuite des approches in silico et in vitro nous ont permis de comprendre les mécanismes moléculaires sous-jacents à la reconnaissance des séquences E-box des promoteurs de gènes cibles par l'héterodimère TE et leur stabilisation sur l'ADN (bouard et al., 2016). Nous avons décrit trois états différents du complexe TWIST1/E12 lié à l'ADN à des séquences E-box fonctionnels et modifiés en fonction de l'affinité de reconnaissance des E-box par TWIST1 / E12 (Bouard et al, 2016). Et enfin, cette approche in silico nous a permis de montrer l’impact de la phosphorylation de TWIST1 sur la dimérisation et sur la liaison à l’ADN des complexes de TWIST1. Cette dernière étude met en exergue l’importance des régulations post-traductionnelles dans l’étude de l’activité de la protéine TWIST1 et dans la recherche d’inhibiteurs / Aberrant reactivation embryonic TWIST1 gene has been identified in the laboratory as a recurrent inactivation mechanism dependent pathways of p53 and Rb in many tumors. Decreasing senescence and the induction of apoptosis, TWIST1 cooperates with oncogenic proteins in cell transformation in vitro and is involved in tumor initiation and progression in vivo. As TWIST1 is very weakly expressed in most adult differentiated cells, it is an attractive target for future therapies. Recently, the heterodimer TWIST1 / E2A (E12 or E47, two alternative splice product of the TCF3 gene) plays a pro-metastatic role in prostate cancer (Gajula et al., 2015), while the complex is the oncogenic form of TWIST1 in human mammary epithelial cells (Jacqueroud et al., 2016). The heterodimerization through HLH areas is a prerequisite for DNA binding domain of training (Massari and Murre 2000). An in silico approach that based homology modeling of complex TWIST1 highlights the key role of inter-hélicales loops in the DNA TWIST1 complex retention student TWIST1 several insertions observed in patients Saethre syndrome -Chotzen (Bouard et al., 2014). Then approaches in silico and in vitro have enabled us to understand the molecular mechanisms underlying the recognition of E-box sequences of the promoters of target genes by TE heterodimer and stabilize DNA (Bouard et al. 2016). We have described three different states of the complex TWIST1 / E12 bound to DNA to functional E-box sequences and modified according to the affinity recognition by E-box TWIST1 / E12 (Bouard et al, 2016). Finally, this approach in silico allowed us to show the impact of TWIST1 phosphorylation on dimerization and binding to DNA TWIST1 complexes. This latest study highlights the importance of post-translational regulation in the study of the activity of the protein and TWIST1 in the search for inhibitors

The binding of different proteins involved in processes such as transcription, replication and chromatin compaction to regions of the genome is regulated by the structure of DNA. Thus, DNA structure acts as the crucial link modulating evolutionary selection of the DNA sequence based on its own function, and the function of the proteins it encodes. The aim of this work is to examine the role of intrinsic, sequence-dependent structural variations vis-a -vis the protein-induced variations, in allowing DNA to assume geometries necessary for binding by proteins. For this purpose, we carried out analyses of datasets of X-ray crystal structures of free and protein-bound DNA, and molecular dynamics simulation studies of few free DNA structures and a protein-DNA complex. Each of the projects described below will appear as a separate chapter in the thesis. Analysis of X-ray crystal structure datasets Dataset of high-resolution X-ray crystal structures of free and protein-bound DNA This project was initiated with the aim of investigating the variation in A-and B-forms of DNA and the role they play in the binding of proteins. However, a survey of the existing literature indicated that the terms ‘A-DNA’ and ‘B-DNA’ were being used rather loosely and several different parameters at the local structural level were being used by various investigators to characterise these structures. Hence a systematic study was taken up to analyse all high-resolution free DNA structures comprising of sufficient number of contiguous Watson-Crick basepairs, irrespective of how they were classified by the existing databases. We also carried out a study of double-helical, Watson-Crick basepaired, free RNA structures for comparison. The structures in the RNA dataset were observed to rigidly assume the A-form and hence the average values of different parameters for that dataset were used to characterise the A-form. The analysis of free DNA and RNA structures was accompanied by an analysis of protein-bound DNA crystal structures. DNA structures bound to the helix-turn-helix motif in proteins were also analysed separately. The analysis of free DNA and RNA structures allowed us to pinpoint the parameters suitable for discriminating A-and B-forms of DNA at the local structural level. The results illustrated that the free DNA molecule, even in the crystalline state, samples a large amount of conformational space, encompassing both the A-and the B-forms. Most protein-bound DNA structures, including those with large, smooth curvature, were observed to assume the B-form. The A-form was observed to be limited to a small number of dinucleotide steps in DNA structures bound to the proteins belonging to a few specific families. Thus our study highlighted the structural versatility of B-form DNA, which allowed it to take up a range of global geometries to accommodate most DNA-binding protein motifs. Dataset of X-ray crystal structures of the nucleosome The study of high-resolution structures of free and protein-bound DNA was followed by an analysis of a dataset of X-ray crystal structures of the nucleosome, which is the fundamental repeating unit of the eukaryotic chromosome, and has been shown to play an important role in transcription regulation. Our results indicated that there is an ensemble of dinucleotide and trinucleotide level parameters that can give rise to similar global nucleosome structures. We therefore raise doubts about the use of the best resolved nucleosome structure as the template to calculate the energy required by putative nucleosome-forming sequences for adopting the nucleosome structure. Based on our results, we have proposed that the local and global level structural variability of DNA may act as a significant factor influencing the formation of nucleosomes in the vicinity of high-plasticity genes, and in determining the probability of binding by regulatory proteins. Molecular dynamics simulation studies of free and protein-bound DNA structures The analysis of crystal structure databases was complemented by molecular dynamics (MD) studies to investigate the dynamic evolution of the DNA structure in its free and protein-bound states. The following three simulation studies were carried out: Study to examine the biological relevance of the presence of 5-methyl group in thymine nucleotides An investigation of the biological relevance of the 5-methyl group in thymine nucleotides was carried out. For this purpose, comparison of molecular dynamics studies on structures with sequences d(CGCAAAUUUGCG)2and d(CGCAAATTTGCG)2was carried out. Our results showed that the presence of the thymine 5-methyl group was necessary for the A-tract to assume characteristic properties such as a narrow minor groove. It was also shown to modulate local level structural parameters and consequently, the curvature of the longer DNA fragment in which the A-tract was embedded. The analysis also provided possible explanation for the experimentally observed interaction of A-tracts with drugs and DNase-I in the presence and the absence of the thymine 5-methyl group. This project was the first of a series of MD studies, and hence several protocols were tested before finalising the correct protocol. Simulations were carried out using the Berendsen temperature equilibration scheme as well as the Langevin temperature equilibration scheme on both the structures. The Langevin temperature equilibration scheme was found to be unsuitable for nucleic acid simulations, as it caused long-term and possibly permanent disruption of the double-helical structure at the terminal and the neighbouring two positions in the sequence. The Berendsen temperature equilibration scheme was not observed to cause such disruptions. Simulations were also carried out on both structures, with or without initialising the initial ion positions. The position of minimum electrostatic potential, where AMBER8 placed the first counterion, was observed to act as a minimum energy trap from which the counterion could not escape even during the course of several nanoseconds of simulation. Hence, the actual simulations were carried out using the Berendsen temperature equilibration scheme, and after randomisation of initial ion positions. The results of protocol testing have been reported in an appendix. Study of DNA bending and curvature An analysis of DNA bending and curvature was carried out, by MD simulation on structures of three, ∼thirty basepair long sequences, namely, d(G-3(CA4T4G)-C)2, d(G-3(CT4A4G)-C)2and d(T-GACTA5T-GACTA6T-GACTA5T-G). For each molecule, snapshots belonging to a particular global geometry (linear, curved, bent in a particular direction etc.) were grouped together, and the average values of the dinucleotide step parameters for different groups were compared. It was observed that for all the three molecules, the average values for groups corresponding to different global geometries were within 1of each other, indicating that ensemble average values of dinucleotide level parameters are incapable of predicting the global geometry of a DNA molecule. Study of the TraR-Trabox complex The study on DNA bending and curvature was followed by simulations of a protein-DNA complex comprising of the bacterial quorum sensing transcription factor TraR with its promoter region known as Trabox. Simulations of a protein-free wild-type Trabox and a Trabox with two mutations in the spacer region were also carried out. Grouping of DNA snapshots in all the three simulations based on average values of dinucleotide parameters in the spacer region shows how selection of the ‘right’ DNA geometry by proteins works at several levels. The number of snapshots of free mutated Trabox assuming a geometry favourable for protein-binding in terms of average twist alone are less than one-fourth of the corresponding number for free wild-type Trabox. When one applies further selection criteria in terms of other parameters such as roll and slide, the number of mutated Trabox snapshots with a geometry favourable for protein-binding drops to less than 0.5%ofthe total number of MD snapshots. Thus our results highlight how sequence-dependent changes in the structrure of DNA regions, adjacent to those that directly hydrogen-bond to proteins, can also critically influence processes such as transcription. General Conclusion Overall, our results indicate that intrinsic, sequence-dependent structural variations in free B-DNA allow it to sample a large volume of the double-helical conformational space, and assume global geometries that can accomodate most DNA-binding proteins.

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