In this thesis, on the basis of the asymmetrical charge distribution of E. coli topoisomerase I, I developed a new rapid procedure to purify E. coli DNA topoismoerase I in the milligram range. The new procedure includes using both cation- and anion-exchange columns, i.e., SP-sepharose FF and Q-sepharose FF columns. E. coli topoisomerase I purified here is free of nuclease contamination. The kinetic constants of the DNA relaxation reaction of E. coli DNA topoisomerase I were determined as well. I also used isothermal titration calorimetry to investigate the energetics of DNA supercoiling by using the unwinding properties of DNA intercalators, ethidium and daunomycin. After comparing the enthalpy changes of these DNA intercalators binding to supercoiled and nicked or relaxed plasmid DNA pXXZ06, I determined the DNA supercoiling enthalpy is about 12 kcal/mol per turn of DNA supercoil, which is in good agreement with the previously published results.

A principle challenge of modern biology is to understand how the human genome is organised and regulated within a nucleus. The field of chromatin biology has made significant progress in characterising how protein and DNA modifications reflect transcription and replication state. Recently our lab has shown that the human genome is organised into large domains of altered DNA helical twist, called DNA supercoiling domains, similar to the regulatory domains observed in prokaryotes. In my PhD I have analysed how the maintenance and distribution of DNA supercoiling relates to biological function in human cells. DNA supercoiling domains are set up and maintained by the balanced activity of RNA transcription and topoisomerase enzymes. RNA polymerase twists the DNA, over-winding in front of the polymerase and under-winding behind. In contrast topoisomerases relieve supercoiling from the genome by introducing transient nicks (topoisomerase I) or double strand breaks (topoisomerase II) into the double helix. Topoisomerase activity is critical for cell viability, but the distribution of topoisomerase I, IIα and IIβ in the human genome is not known. Using a chromatin immunoprecipitation (ChIP) approach I have shown that topoisomerases are enriched in large chromosomal domains, with distinct topoisomerase I and topoisomerase II domains. Topoisomerase I is correlated with RNA polymerase II, genes and underwound DNA, whereas topoisomerase IIα and IIβ are associated with each other and over-wound DNA. This indicates that different topoisomerase proteins operate in distinct regions of the genome and can be independently regulated depending on the genomic environment. Transcriptional regulation by DNA supercoiling is believed to occur through changes in gene promoter structure. To investigate DNA supercoiling my lab has developed biotinylated trimethylpsoralen (bTMP) as a DNA structure probe, which preferentially intercalates into under-wound DNA. Using bTMP in conjunction with microarrays my lab identified a transcription and topoisomerase dependent peak of under-wound DNA in a meta-analysis of several hundred genes (Naughton et al. (2013)). In a similar analysis, Kouzine et al. (2013) identified an under-wound promoter structure and proposed a model of topoisomerase distribution for the regulation of promoter DNA supercoiling. To better understand the role of supercoiling and topoisomerases at gene promoters, a much larger-scale analysis of these factors was required. I have analysed the distribution of bTMP at promoters genome wide, confirming a transcription and expression dependent distribution of DNA supercoils. DNA supercoiling is distinct at CpG island and non-CpG island promoters, and I present a model in which over-wound DNA limits transcription from both CpG island promoters and repressed genes. In addition, I have mapped by ChIP topoisomerase I and IIβ at gene promoters on chromosome 11 and identified a different distribution to that proposed by Kouzine et al. (2013), with topoisomerase I maintaining DNA supercoiling at highly expressed genes. This study provides the first comprehensive analysis of DNA supercoiling at promoters and identifies the relationship between supercoiling, topoisomerase distribution and gene expression. In addition to regulating transcription, DNA supercoiling and topoisomerases are important for genome stability. Several studies have suggested a link between DNA supercoiling and instability at common fragile sites (CFSs), which are normal structures in the genome that frequently break under replication stress and cancer. bTMP was used to measure DNA supercoiling across FRA3B and FRA16D CFSs, identifying a transition to a more over-wound DNA structure under conditions that induce chromosome fragility at these regions. Furthermore, topoisomerase I, IIα and IIβ showed a pronounced depletion in the vicinity of the FRA3B and FRA16D CFSs. This provides the first experimental evidence of a role for DNA supercoiling in fragile site formation.

Ganoderma lucidum is a popular herbal medicine used in China to promote health. Modern studies have disclosed that the active ingredients of Ganoderma can exhibit several effects, including antitumor effects and immunomodulation. The present study evaluated the antitumor effects of self-prepared Ganoderma extracts and spores oil, and investigated the possible underlying mechanisms by observing the effects of the extracts and oil on topoisomerases and the cell cycle. The results showed that Ganoderma extracts and spores oil presented dose-dependent inhibitory effects on tumor cells. The half maximal inhibitory concentration (IC50) values of Ganoderma extracts on HL60, K562 and SGC-7901 cells for 24 h were 0.44, 0.39 and 0.90 mg/ml, respectively; for Ganoderma spores oil, the IC50 values were 1.13, 2.27 and 6.29 mg/ml, respectively. In the in vivo study, the inhibitory rates of Ganoderma extracts (4 g/kg/d, intragastrically) on S180 and H22 cells were 39.1 and 44.6%, respectively, and for Ganoderma spores oil (1.2 g/kg/d, intragastrically) the inhibitory rates were 30.9 and 44.9%, respectively. Ganoderma extracts and spores oil inhibited the activities of topoisomerase I and II. Ganoderma spores oil was shown block the cell cycle at the transition between the G1 and S phases and induce a marked decrease in cyclin D1 levels in K562 cells, with no significant change in cyclin E level. These results suggest that the Ganoderma extracts and spores oil possessed antitumor effects in the in vitro and in vivo studies. The antitumor mechanisms of the extracts and spores oil were associated with inhibitory effects on topoisomerase I and II activities, and for Ganoderma spores oil, the antitumor effects may also be associated with decreased cyclin D1 levels, thus inducing G1 arrest in the cell cycle.

La famille des GTPases Rho, comprenant 20 membres, contrôle la dynamique du cytosquelette d'actine et différents processus cellulaires comme la migration. En plus de leurs rôles bien établis, certaines GTPases Rho, notamment RhoB et Rac1, ont émergé en tant que gènes de réponse précoce aux dommages à l'ADN. En effet, RhoB est induite en réponse à divers stress génotoxiques, y compris la camptothécine (CPT), les UV et le cisplatine, et protège principalement les cellules de l'apoptose. Le rôle des autres GTPases Rho en réponse précoce aux génotoxiques reste largement méconnu. Dans ce projet, nous avons utilisé la camptothécine, un inhibiteur de la topoisomérase I (TOP1), qui stabilise sélectivement les complexes de clivage TOP1-ADN (TOP1cc) sur la chromatine, afin de cribler les GTPases Rho induites de façon précoce par les dommages à l'ADN. En plus de RhoB, nous avons identifié RND1 comme un gène rapidement induit par la CPT. L'induction de RND1 est réversible et étroitement corrélée à la présence de TOP1cc induit par la CPT. En accord avec ces observations, les rayons UV et le péroxyde d'hydrogène, qui stabilisent indirectement les TOP1cc, induisent également RND1. La CPT augmente la transcription de RND1 indépendamment de l'activité de son promoteur minimal. De plus, la CPT augmente l'activité de la poly ADP-ribose polymérase (PARP1), dont l'inhibition prévient la transcription de RND1. La surexpression de RND1 augmente également l'expression de PARP1, suggérant une régulation positive entre PARP1 et RND1 en réponse aux TOP1cc. Ainsi, nous proposons qu'en réponse à la CPT, les TOP1cc activent PARP1, qui à son tour favorise la transcription de RND1, initiant ainsi une boucle de rétrocontrôle positive. Enfin, nous avons montré que RND1 protège les cellules contre l'apoptose induite par la CPT et entraîne leur résistance à la CPT. L'ensemble de ces résultats ont permis d'identifier RND1 comme nouvelle GTPase Rho impliquée dans la réponse au stress et proposent un nouveau mécanisme de régulation de la transcription des gènes en réponse aux TOP1cc via l'activation de PARP1. Ces résultats suggèrent par ailleurs qu'inhiber la signalisation de RND1 pourrait sensibiliser les cellules tumorales aux dérivés cliniques de la CPT. / Rho GTPase family comprises 20 members that regulate key cellular functions such as actin cytoskeleton organization and migration. Beside their canonical functions, certain Rho GTPases, including RhoB and Rac1, emerged as early DNA damage-inducible genes. Indeed, RhoB is readily induced in response to various genotoxic stress, including camptothecin (CPT), UV and cisplatin, and primarily protect cells against apoptotic cell death. Whether other Rho GTPases also respond early to genotoxics is largely unknown. In this project, we used camptothecin, a topoisomerase I (TOP1) inhibitor that selectively stabilized TOP1-DNA cleavage complexes (TOP1cc) onto chromatin, to screen for early DNA damage-inducible Rho GTPases. Besides RhoB, we identified RND1 as a gene rapidly induced by CPT. RND1 induction is reversible and closely associated with the presence of TOP1cc induced by CPT. Consistently, UV light and hydrogen peroxide, which indirectly stabilized TOP1cc, induce RND1 as well. CPT increases minimal promoter-independent RND1 transcription. Additionally, CPT increases poly ADP-ribose polymerase (PARP1) activity, whose inhibition prevents RND1 transcription. Overexpression of RND1 also increases PARP1 expression, suggesting a positive regulation between PARP1 and RND1 in response to TOP1cc. Thus, we propose that in response to CPT, TOP1cc activate PARP1, which in turn promotes RND1 transcription resulting in a positive feedback loop. Finally, we found that RND1 protects cells against CPT-induced apoptosis and leads to resistance to CPT. Together, these results highlight RND1 as a new Rho GTPase involved in the response to stress and propose a new mechanism for TOP1cc-induced gene transcription through PARP1 activation. These findings further suggest that inhibiting RND1 signaling could sensitize tumor cells to CPT derivatives.

The thesis entitled “Benzimidazole based Novel Ligands for Specific Recognition of Duplex and G-Quadruplex DNA” deals with the design, synthesis and modeling of several benzimidazole based molecules and their interaction with duplex and G-quadruplex DNA structures. It also elucidates the inhibition effect of the ligands on the activity of Topoisomerase I and Telomerase. The work has been divided into six chapters. Chapter 1. DNA Interacting Small Organic Molecules: Target for Cancer Therapy This first chapter presents an overview on the various types of small molecules that interact with duplex and G-quadruplex structures of DNA or interfere with the activity of DNA targeted enzymes like topoisomerase and telomerase. The importance of such molecules as chemotherapeutic agents is highlighted. Chapter 2. DNA Recognition: Conformational Switching of Duplex DNA by Mg2+ ion Binding to Ligand Bis-benzimidazoles like Hoechst 33258 are well known ligands that bind to duplex DNA (ds-DNA) minor grooves. Here a series of dimeric bisbenzimidazole based ligands in which two Hoechst units are connected via oxyethylene based hydrophilic [Ho-4ox-Ho (1), Ho-3ox-Ho (2)] or via hydrophobic oligomethylene [Ho-(CH2)8-Ho (3)](Figure 1) spacers have been synthesized. The aim of this investigation is to examine the binding property of these dimers on the ds-DNA to explore whether the variation in the length of the spacer has any effect on DNA binding properties particularly in presence of selected metal ions. The changes of individual dimers in DNA binding efficiency was studied in detail by fluorescence, circular dichroism spectral titrations and thermal denaturation experiment with selected duplex DNA formed from appropriate oligonucleotides. We have also examined the changes that occur in geometry of the molecules from linear to hairpin motif in presence of Mg2+ ion. A large difference was observed in [ligand]/ [DNA] ratio and binding efficiency with ds-DNA upon change in the ligand geometry from linear to hairpin motif. The experimental results were then substantiated using docking and molecular dynamics simulations using a model ds-DNA scaffold. Both experimental and theoretical studies indicate that the DNA binding is highly dependent on the spacer type and length between the two monomeric Hoechst units. The spacer length actually helps to achieve shape complimentarity with the double-helical DNA axis. Figure1: Chemical structures of the dimeric ligands Ho-4ox-Ho, Ho-3ox-Ho, Ho-(CH2)8-Ho and Hoechst 33258 (Ho) used in this study. Chapter 3. DNA Binding and Topoisomerase I Inhibiting Properties of New Benzimidazole Substituted Polypyridyl Ruthenium (II) Mixed-Ligand Complexes In this study, we have synthesized and fully characterized three new Ru(II) based polypyridyl and benzimidazole mixed complexes: (1) [Ru(bpy)2(PMI)], 2+ (2) [Ru(bpy)2(PBI)]2+ and (3) [Ru(bpy)2(PTI)]2+ (Figure 2) . The affinities of these complexes toward duplex DNA were investigated. In addition, the photocleavage reaction of DNA and topoisomerase I inhibition properties of these metal complexes were also studied. The DNA binding efficiency of individual complexes was studied in detail by absorbance, fluorescence spectral titrations and thermal denaturation experiment using natural calf-thymus DNA. Upon irradiation at 365 nm, all three Ru(II) complexes were found to promote the cleavage of plasmid DNA from negatively supercoiled to nicked circular and subsequently to linear DNA. The inhibition of topoisomerase I mediated by these Ru(II) complexes was also examined. These experiments demonstrate that each complex serves as an efficient inhibitor toward topoisomerase I and such inhibition activity is consistent with interference with the DNA religation step catalyzed by topoisomerase. Figure 2. Chemical structures of the metal complexes used in this present study. Chapter 4. Synthesis and Evaluation of a Novel Class of G-Quadruplex-Stabilizing small molecules based on the 1,3-Phenylene-bis (piperazinyl benzimidazole) syatem Achieving stabilization of telomeric DNA in the G-quadruplex conformation by various organic compounds is an important goal for the medicinal chemists seeking to develop new anticancer agents. Several compounds are known to stabilize the G-quadruplexes. However, relatively few are known to induce their formation and/or alter the topology of the pre-formed G-quadruplex DNA. Herein, four compounds having the 1,3-phenylene-bis(piperazinyl benzimidazole) (Figure 3) unit as a basic skeleton have been synthesized, and their interactions with the 24-mer telomeric DNA sequences from Tetrahymena thermophilia d(T2G4)4 have been investigated using high-resolution techniques such as circular dichroism (CD) spectropolarimetry, CD melting, emission spectroscopy, and polyacrylamide gel electrophoresis. The data obtained, in the presence of one of three ions (Li+, Na+ or K+), indicate that all the new compounds have a high affinity for G-quadruplexDNA, and the strength of the binding with G-quadruplex depends on (i) phenyl ring substitution, (ii) the piperazinyl side chain, and (iii) the type of monovalent cation present in the buffer. Results further suggest that these compounds are able to abet the conversion of the intramolecular G-quadruplex DNA into parallel stranded intermolecular G-quadruplex DNA. Notably, these compounds are also capable of inducing and stabilizing the parallel stranded G-quadruplex DNA from randomly structured DNA in the absence of any stabilizing cation. The kinetics of the structural changes induced by these compounds could be followed by recording the changes in the CD signal as a function of time. Figure 3. Chemical structures of the ligands used in this study. Chapter 5A. The Spacer Segment in the Dimeric 1,3-phenylene-bis (piperazinyl benzimidazole) has a Dramatic Influence on the Binding and Stabilization of Human Telomeric G-Quadruplex DNA Ligand-induced stabilization of G-quadruplex structures formed by human telomeric DNA is an active area of basic and clinical research. The compounds which stabilize the G-quadruplex structures lead to suppression of telomerase activity. Herein, we present the interaction of a series of monomeric and dimeric compounds having 1,3-phenylene-bis(piperazinyl benzimidazole) (Figure 4) as basic pharmacophore unit with G-quadruplex DNA formed by human telomeric repeat d[(G3T2A)3G3]. These new compounds provide an excellent stabilization property to the pre-formed G-quadruplex DNA in the presence of one of three ions (100 mM Li+, Na+ or K+ ions). Also the G-quadruplex DNA formed in the presence of low concentrations of ligands in 100 mM K+, adopts a parallel-stranded conformation which attains an unusual thermal stability. The dimeric ligands having oxyethylene based spacer provide much higher stability to the pre-formed G-quadruplex DNA and the G-quadruplexes formed in presence of the dimeric compounds than the corresponding monomeric counterparts. Consistent with the above observation, the dimeric compounds exert significantly higher telomerase inhibition activity than the monomeric compounds. The ligand induced G-quadruplex DNA complexes were further investigated by computational molecular modeling, which provide useful information on their structure-activity relationship. Figure 4. Chemical structures of the monomeric and dimeric ligands used in this study. Chapter 5B. Role of Spacer in Symmetrical Gemini bisbenzimidazole based Ligands on the Binding and Stabilization of Dimeric G-Quadruplex DNA derived from Human Telomeric Repeats The design and development of anticancer agents that act via stabilization of the telomeric G-quadruplex DNA is an active area of research because of its importance in the negative regulation of telomerase activity. Several classes of G-quadruplex DNA binding ligands have been developed so far, but they mainly act on the DNA sequences which are capable of forming a single Gquadruplex unit. In the present work, we have developed few new dimeric (Gemini) bisbenzimidazole ligands (Figure 5), in which the spacer joining the two bisbenzimidazole units have been varied using oligooxyethylene units of different length. Herein we show the interaction of each of these ligands, with the G-quadruplex DNA, derived from oligodeoxynucleotides d(T2AG3)4 and d(T2AG3)8, which fold into a monomeric and dimeric (having two folded G-tetrad units) G-quadruplex DNA, respectively. We also present evidence that the G-quadruplex DNA structure formed by these sequences in K+ solution in presence of the ligands is parallel, with unusual stability, and the spacer length between the two bisbenzimidazole units has critical role on the G-quadruplex stability, particularly on the G-quadruplex structures formed by the 48-mer sequence. The computational aspects of the ligand-G-quadruplex DNA association have also been analyzed. Interestingly, the gemini ligand having longer spacer was highly potent in the inhibition of telomerase activity than the corresponding gemini ligands having shorter spacer or the monomeric ligand. Also, the dimeric ligands are more cytotoxic toward the cancer cells than normal cells. Figure 5. Chemical structures of the monomeric and gemini ligands used in this study. Chapter 6. Stabilization and Structural Alteration of G-Quadruplex DNA made from Human Telomeric Repeat Mediated by Novel Benzimidazole Derivatives based on Tröger’s Base Ligand-induced stabilization of G-quadruplex formation by the telomeric DNA single stranded 3'-overhang is a nice strategy to inhibit telomerase from catalyzing telomeric DNA synthesis and form capping telomeric ends. Herein we present the first report of the interactions of two novel bisbenzimidazoles (TBBz1 and TBBz2)(Figure 6) based on the Tröger’s base skeleton with the G-quadruplex DNA. These molecules stabilize the G-quadruplex DNA derived from a human telomeric sequence. Significantly strong binding affinity of these molecules to G-quadruplex DNA relative to duplex DNA was observed by CD spectroscopy, thermal denaturation and UV-vis titration studies. The above results obtained are in excellent agreement with the biological activity, measured in vitro using a modified TRAP assay. Additionally exposure of cancer cells to these compounds showed a remarkable decrease in the population growth. Also, it has been observed that the ligands are selectively more cytotoxic toward the cancerous cells than the corresponding noncancerous cells. To understand further, the ligand-G-quadruplex DNA complexes were investigated by computational molecular modeling. This provided additional insights on the structure activity relationship. Computational studies suggest that the adaptive scaffold not only allows these ligands to occupy the G-quartet but also binds with the grooves of the G-quadruplex DNA. Figure 6. Chemical structures of the ligands, TBBz1 and TBBz2 used in this study, (For structural formula pl see the abstact.pdf file.)

No description available.

Protein-protein interactions (PPIs) are essential features of cellular processes including DNA replication, transcription, translation, recombination, and repair. In my study, the protein interactions of bacterial DNA topoisomerase I, an essential enzyme, were investigated. The topoisomerase I in bacteria relaxes excess negative supercoiling on DNA and maintains genomic stability. Investigating the PPI network of DNA topoisomerase I can further our understanding of the various functional roles of this enzyme. My study is focused on topoisomerase I of Escherichia coli and Mycobacterium smegmatis. Firstly, we have explored the biochemical mechanisms for an interaction between RNA Polymerase, and topoisomerase I in E. coli. Molecular docking and molecular dynamic simulations have predicted that the interactions are mediated through electrostatic, and hydrogen bonding. The predicted Lysine residues (K627, K664) of topoisomerase I that are involved in the electrostatic interactions were mutated to Alanine, and its effect on the binding efficiency with RNA polymerase was reported. In a separate study, PPI partners of topoisomerase I in mycobacteria were identified. Knowledge gained from the study can provide valuable insights into the physiological functions of a validated drug target, DNA topoisomerase I, in pathogenic mycobacteria. Co-immunoprecipitation and pull-down assays were coupled to mass spectrometry for identification of the protein partners of mycobacterial topoisomerase I. The study has identified RNA polymerase, and putative helicases (DEAD/DEAH BOX helicases) as potential protein partners of mycobacterial topoisomerase I. My results indicated that the tail region of the CTD-topoisomerase I was required for direct physical interaction with the RNAP beta’ subunit. My studies have also verified the physiological relevance of the topoisomerase I - RNA polymerase interactions for survival under antibiotic, and oxidative stress. Lastly, I report a direct physical interaction between E. coli topoisomerase I and RecA by pull-down assays. Previous studies have shown that RecA, a DNA repair protein, can stimulate the relaxation activity of E. coli topoisomerase I. Our new results showed that the stimulatory effect can be attributed to the physical interaction of topoisomerase I with RecA.

Les protéines SR, qui constituent une famille conservée de facteurs liant l'ARN, jouent un rôle majeur dans l'épissage des ARN et en particulier dans la régulation de l'épissage alternatif. Certaines protéines SR peuvent également participer à l'élongation de la transcription, l'export, la stabilité ou la traduction des ARNm. Ces différents rôles soulignent l'importance des protéines SR en tant que régulateurs clés du métabolisme des ARNm et de l'expression des gènes. Des altérations de leur quantité ou de leur activité peuvent induire des défauts développementaux ou des pathologies telles que des tumeurs. Afin de mieux comprendre les fonctions et les mécanismes de régulation des protéines SR in vivo, je me suis intéressée à la protéine SR B52 chez D. melanogaster. En réalisant un crible génétique, nous avons identifié des protéines capables de sauver les phénotypes induits par la surexpression de B52 in vivo. L'une de ces protéines est l'ADN Topoisomérase I (Topo I). La Topo I possède à la fois une activité topoisomérase impliquée dans la relaxation de l'ADN, et une activité kinase capable de phosphoryler les protéines SR. Nous avons montré que B52 est impliquée dans le recrutement de la Topo I aux sites actifs de transcription, en particulier lors de l'induction des gènes heat shock, et que ces protéines jouent un rôle dans la libération de l'ARN hsp70 de son site de transcription et dans l'extinction de sa transcription. Une autre protéine capable de sauver les phénotypes induits par la surexpression de B52 est Brain tumor, un répresseur post-transcriptionnel de l'expression de myc. Myc est un régulateur clé de la croissance chez la drosophile. Nos résultats révèlent un effet positif de B52 sur la croissance cellulaire dans certains tissus, et sur l'expression de dmyc. Nous montrons également que le niveau de B52 affecte l'épissage alternatif de plusieurs gènes impliqués dans la croissance, dont le coactivateur transcriptionnel Yorkie et le facteur d'initiation de la traduction eIF4E. Ainsi, nos travaux suggèrent que la protéine SR B52 pourrait coordonner un ensemble d'évènements d'épissage dans des voies de signalisation impliquées dans la croissance cellulaire. / SR proteins, which constitute a conserved family of RNA-binding factors, play a key role in RNA splicing and particularly in alternative splicing regulation. In addition, some SR proteins have been shown to participate in transcription elongation, mRNA export, mRNA stability and mRNA translation. These wide-ranging roles of SR proteins highlight their importance as pivotal regulators of mRNA metabolism and gene expression. Alteration of their expression level or activity can induce developmental defects or pathologies such as tumors. To better understand SR proteins functions and how they are regulated in vivo, I studied a major SR protein in Drosophila melanogaster called B52. Using a genetic screen, we identified proteins that can rescue the phenotypes induced by B52 overexpression. Among them is the DNA Topoisomerase I (Topo I). Topo I carries two enzymatic activities: a topoisomerase activity that can relax DNA supercoiling generated by transcription or replication, and a kinase activity which phosphorylates SR proteins. We showed that B52 is required for Topo I recruitment to active transcription sites, especially at the heat shock genes upon their induction, and that these proteins play a role in hsp70 mRNA release from its transcription site and in its transcription shutdown. Another protein that can rescue the phenotypes induced by B52 overexpression is Brain tumor, a post-transcriptional repressor of myc expression. Myc is a major regulator of cell growth in Drosophila. Our results reveal a positive effect of B52 on cell growth in some tissues, and on myc expression. We also show that B52 level can affect the alternative splicing of several genes involved in cell growth, especially that of the transcriptional coactivator Yorkie and the translation initiation factor eIF4E. Thus, our work suggests that the SR protein B52 could coordinate a range of splicing events in signalling pathways involved in cell growth.

La sclérose systémique est une maladie autoimmune dont l’une des complications majeures est la fibrose. La DNA topoisomérase I (topo) est l’un des principaux autoantigènes associés à cette maladie. Toutefois, aucun lien n’a encore pu être établi entre la présence des anti-topo et le développement de la fibrose. Les travaux antérieurs du laboratoire d’accueil ont montré une interaction directe de la topo avec la surface des fibroblastes et la matrice extracellulaire. Nous avons voulu caractériser ces interactions du point de vue moléculaire. La topo a donc été exprimée sous forme de 5 fragments, déterminés à partir de ses principaux domaines structuraux et de ses épitopes majeurs, chez E. coli. Les fragments purifiés ont été analysés pour leur interaction avec l’héparine, représentant les héparane sulfates de la surface des fibroblastes, et avec des protéines purifiées de la matrice extracellulaire. Nous avons montré que le fragment topo-N est le principal responsable de l’interaction avec l’héparine, ce qui suggère donc l’implication potentielle de ce domaine dans l’interaction de la topo avec la surface des fibroblastes. Le fragment topo-DIDII est responsable de l’interaction avec la plupart des protéines de la matrice extracellulaire étudiées, alors que le fragment topo-H15 n’interagit qu’avec la vitronectine. Aucune interaction des fragments topo-DIII et topo-C n’a été décelée. Ces résultats pourront maintenant servir à mieux comprendre le rôle potentiel de la topo et des autoanticorps circulants anti-topo dans la fibrose présente chez les personnes atteintes de sclérose systémique en contribuant à l’identification de la cible de la topo sur les fibroblastes. / Systemic sclerosis is an autoimmune disease in which one of the major complications is fibrosis. DNA topoisomerase I (topo) is a major autoantigen associated with this disease. However, no link has yet been established between the presence of anti-topo and the development of fibrosis. Previous work of the host laboratory showed a direct interaction of the topo with the surface of fibroblasts and extracellular matrix. We wanted to characterize these interactions at the molecular level. Topo was expressed in 5 fragments, determined from its main structural domains and its major epitopes, in E. coli. The purified fragments were analyzed for their interaction with heparin, representing heparan sulfates on the surface of fibroblasts, and with purified proteins of the extracellular matrix. We have shown that the topo-N fragment is responsible for interaction with heparin, suggesting hence, potential involvement of this domain in the interaction of topo with the surface of fibroblasts. The topo-DIDII fragment is responsible for the interaction with most proteins of the extracellular matrix studied, whereas the topo-H15 fragment only binds to vitronectin. No interaction of fragments topo-DIII and topo-C was found. These results can now be used to better understand the potential role of topo and circulating anti-topo autoantibodies in the fibrosis present in patients with systemic sclerosis in helping to identify the target of topo on fibroblasts.

The thesis entitled “Design and Synthesis of Benzimidazole Based Templates in Duplex and Quadruplex DNA Recognition and in Topoisomerase Inhibition” deals with the design and synthesis of several benzimidazole based molecules and their interaction with duplex and quadruplex DNA structures. It also elucidates the inhibition effect of the compounds on the activity of topoisomerase I enzyme of parasitic pathogen Leishmania donovani. The work has been divided into five chapters. Chapter 1: An Introduction to DNA and its Interaction with Small molecules. The first chapter provides an introduction to the double helical structure of DNA and the central dogma that suggests the flow of genetic information from DNA to RNA to protein. This chapter also presents an overview on the various types of small molecules that interact with duplex and quadruplex structures of DNA or interfere with the activity of DNA targeted enzymes like topoisomerase. This chapter describes the importance of such molecules as chemotherapeutic agents. Chapter 2 deals with three isomeric, symmetrical bisbenzimidazole derivatives bearing pyridine on the two termini. The syntheses, duplex DNA binding and computational structure analyses of the molecules have been divided into two sections. Chapter 2A: Novel Symmetrical Pyridine Derivatized Bisbenzimidazoles: Synthesis and Unique Metal Ion Mediated Tunable DNA Minor Groove Binding. The first chapter deals with the synthesis and double stranded (ds) DNA binding characteristics of the three bisbenzimidazole derivatives. Despite being positional isomers, their relative binding affinities towards ds-DNA varied considerably. Fluorescence, circular dichroism and temperature dependent UV-absorption spectroscopy have been employed to characterize ligand-DNA binding interaction. All spectroscopic studies revealed the strong A-T selective DNA binding affinities of the p- and m-pyridine derivatized molecules (p-pyben and m-pyben respectively) and indicated dramatically weak binding interaction of the ortho derivative (o-pyben) to ds-DNA. Additionally, unique transition metal ion mediated tunable DNA binding shown by o-pyben has been described in this chapter. While the ds-DNA binding characteristics of p- and m-pyben remained unaffected in presence of metal ions, that of o-pyben could be reversibly ‘switched off’ in the presence of divalent transition metal ions like Co2+, Ni2+, and Cu2+. Addition of EDTA reversed the effects and DNA binding was again observed. This interesting observation provides valuable insight into the DNA recognition property of these isomeric bisbenzimidazole derivatives. Figure 1. Molecular structures of pyridine derivatized symmetrical bisbenzimidazoles. Chapter 2B: Differential Binding of Positional Isomers of Symmetric Bisbenzimidazoles on DNA Minor-Groove: A Computational study. To explain the weak DNA binding affinity of o-pyben, compared to p- or m-pyben, detailed ab initio/DFT computational analyses of the inherent structural features of the three isomers were performed both in the gas-phase and in water. The study revealed the presence of intramolecular hydrogen bond existing in the opyben, between the benzimidazole proton (H3) and the pyridine nitrogen (N1). Additionally, potential energy scans for rotation about the bonds connecting the pyridine-benzimidazole and benzimidazole-benzimidazole fragments were performed. This revealed surprising conformational rigidity existing in the o- isomer that resisted any out-of-plane twisting of the pyridine-benzimidazole fragment. The presence of intramolecular H-bonding was further confirmed by experimental determination of pKa of the three isomers. The molecules being bisbenzimidazole derivatives bound to the minor groove of ds-DNA, the benzimidazole protons forming hydrogen bonded interactions with the DNA bases. However in the o- derivative, the intramolecular hydrogen bonding made the crucial benzimidazole protons unavailable for DNA binding thereby leading to its poor interaction with DNA. Chapter 3. Novel Series of Anthra[1,2-d]imidazole-6,11-dione Derivatives: Synthesis, DNA Binding and Inhibition of Topoisomerase I of Leishmania donovani This chapter describes the synthesis of nine imidazole fused anthraquinone derivatives and their interaction with double-stranded DNA, investigated by UV-visible absorption spectroscopy and viscometric titrations. Figure 2. Molecular structures of the imidazole fused anthraquinone derivatives. All the molecules showed intercalative mode of binding to double stranded DNA, though their relative binding affinities were different. Next their inhibitory effects on the catalytic activity of topoisomerase I enzyme of Leismania donovani were investigated. L. donovani is the causative agent for human visceral leishmaniasis; a fatal disease affecting liver and spleen. Five out of the nine derivatives tested, proved to be extremely efficient inhibitors of the enzyme. Of them, three showed greater inhibition potency than camptothecin, a well-established topoisomerase I inhibitor and the precursor for several clinically useful anti-tumor drugs. The molecules were shown to inhibit by the stabilization of enzyme-DNA cleavable complex, and the inhibition efficiency was found to be highly dependent on the pKa of the side-chain nitrogen. These results provide useful insights towards developing more potent inhibitors of the parasitic enzyme. As the compounds are synthetically facile, chemically stable and possess long shelf life, they should be attractive candidates for design of novel family of topoisomerase I inhibitor. Indeed the nature of amine based side chain and its pKa would hold the key in such design. Chapter 4 deals with a series of symmetrical bisbenzimidazole derivatives in which the benzimidazole units have been connected via different aromatic linkers. The syntheses, duplex DNA interaction, topoisomerase inhibition and quadruplex DNA stabilization shown by these four molecules have been divided into two sections. Chapter 4A. Synthesis, Duplex DNA Binding and Topoisomerase I Inhibition by Symmetrical Bisbenzimidazole Derivatives with Aromatic Linkers. This chapter describes the synthesis of four symmetrical bisbenzimidazole derivatives bearing aromatic linkers, phenyl, naphthyl or anthryl between the benzimidazole rings. Next their interaction with duplex DNA was investigated using fluorescence and temperature dependent UV absorption spectroscopy and viscometric titration techniques. Addition of DNA caused fluorescence enhancement of the molecules implying their interaction with duplex DNA. All the four molecules on binding to double helical DNA induced thermal stabilization of the latter. Viscometric titration of calf thymus DNA with the four compounds revealed a partial-intercalative mode of binding for the anthracene derivatized molecule 4. Next, their inhibitory effects on the catalytic activity of topoisomerase I enzyme were studied. The anthracene derivatized compound (4) showed high inhibition of the enzyme catalyzed relaxation of supercoiled plasmid DNA. Naphthalene derivatized compound (3) exhibited weak inhibition whereas the derivatives bearing 1,4- and 1,3-disubstitued benzene (1 and 2 respectively) units showed no inhibition. Figure 3. Molecular structures of the symmetrical bisbenzimidazole derivatives. Chapter 4B. Quadruplex DNA Stabilization by Symmetrical Bisbenzimidazole Derivatives with Aromatic Linkers. The ability of the aforementioned molecules to stabilize G-quadruplex structures was investigated next. DNA quadruplex secondary structures are potential molecular targets for new generation chemotherapeutic drugs; hence there is an impetus in developing quadruplex targeting molecules. The Tetrahymena thermophilia telomeric sequence 5´-(T2G4)4-3´ was selected for the studies as it exhibits interesting structural polymorphism depending on whether quadruplex formation occurs in presence of Na+ or K+. Circular dichroism and fluorescence anisotropy techniques were used to study the interaction of these newly synthesized molecules with quadruplex DNA. Also thermal stabilization of quadruplex structure induced by the molecules was determined by temperature dependent UV absorption studies. The compounds 1, 3 and 4 stabilized Na+ induced quadruplex without causing any structural alterations of the latter. However, the m-phenyl linker bearing molecule 2, above a certain [ligand]/[DNA] concentration ratio, caused uniquestructural alteration of the Na+ induced quadruplex such that the CD-signature of the latter resembled that of a K+ induced quadruplex structure. This result was corroborated by quadruplex thermal melting data and fluorescence anisotropy. Interestingly this ligand was also able to induce secondary structure formation in randomly oriented ss-DNA, akin to K+ induced quadruplex structure, even in the absence of Na+ or K+. Chapter 5. Synthesis and DNA Binding of Novel Biscationic Dimers of Bisbenzimidazole Systems. This chapter describes the design, synthesis and ds-DNA binding properties of four dicationic dimers of bisbenzimidazoles. Targeting long base pair sequences in double helical DNA is a key issue in chemical biology and connecting different DNA binding modules by appropriate linkers is an attractive strategy for achieving the same. The precursor monomer unit was a bisbenzimidazole derivative and an analogue of Hoechst 33258. Two such moieties were connected via bisoxyethylenic or 6- or 3-methylenic or piperazinyl units to achieve linker of varying length, rigidity and hydrophilicity. To study the interaction of the dimers with duplex DNA, fluorescence and circular dichroism spectroscopy were used. Two of the dimers, (bbim-2ox-bbim and bbim-6met-bbim) bearing long flexible spacers, were able to target 13-AT base pairs long oligonucleotide sequences in a 1:1 binding mode with an affinity 8-10 times better than the precursor monomer or Hoechst 33258. Also thermal denaturation experiments showed high duplex stabilization induced by the same two dimers. All studies indicated a bidentate mode of binding where both the arms of the dimers participated in DNA binding. The molecules bearing the short and rigid linkers (bbim-3met-bbim and bbimpiper- bbim) on the other hand showed low binding affinity towards duplex DNA, as indicated by fluorescence, circular dichroism and thermal melting studies. The short linkers probably did not favor simultaneous binding of both the monomeric arms of the dimers to DNA minor groove. The work reported in this chapter indicates the strong influence of the length and nature of linker in determining drug/DNA binding affinity. Figure 4. Molecular structures of dicationic dimeric bisbenzimidazole derivatives.(Refer PDF File)