In this thesis, emphasis has been placed on development of new approaches for high-throughput analysis of protein:DNA interactions in vitro and in vivo. In vitro strategies for detection of protein:DNA interaction require isolation of active and soluble protein. However, current methodologies for purification of proteins often fail to provide high yield of pure and tag-free protein mainly because enzymatic cleavage reactions for tag removal do not exhibit stringent sequence specificity. Solving this problem is an important step towards high-throughput in vitro analysis of protein:DNA interactions. As a result, parts of this thesis are devoted to developing new approaches to enhance the specificity of a proteolysis reaction. The first approach was through manipulation of experimental conditions to maximize the yield of the desired protein products from enterokinase proteolysis reactions of two His-tagged proteins. Because it was suspected that accessibility of the EK site was impeded, that is, a structural problem due to multimerization of proteins, focus was based on use of denaturants as a way to open the structure, thereby essentially increasing the stoichiometry of the canonical recognition site over noncanonical, adventitious sites. Promoting accessibility of the canonical EK target site can increase proteolytic specificity and cleavage yield, and general strategies promoting a more open structure should be useful for preparation of proteins requiring endoprotease treatment. One such strategy for efficient EK proteolysis is proposed: by heterodimerizing with a separate leucine zipper, the bZIP basic region and amino-terminus can become more open and potentially more accessible to enterokinase. In vivo strategies have the advantage over their in vitro counterparts of providing a native-like environment for assessing protein:DNA interactions, yet the most frequently used techniques often suffer from high false-positive and false-negative rates. In this thesis, a new bioprobe system for high-throughput detection of protein:DNA interactions in vivo is presented. This system offers higher levels of accuracy and sensitivity as well as accessibility and ease of manipulation in comparison with existing technologies.

Regulation of transcription initiation is the major determining event employed by the cell to control gene expression and subsequent cellular processes. The weak promoters, with low basal transcription activities, are activated by activators. Bacteriophage Mu mom gene, which encodes a unique DNA modification function, is detrimental to cell when expressed early or in large quantities. Mu has designed a complex, well-controlled and orchestrated regulatory network for mom expression to ensure its synthesis only in late lytic cycle. The phage encoded transcription activator protein C activates the gene by promoter unwinding of the DNA and thereby recruiting of RNAP to the promoter. C protein functions as a dimer for DNA binding and transcription activation. Mutagenesis and chemical crosslinking studies revealed that the leucine zipper motif, and not the coiled coil motif in the N terminal region, is responsible for C dimerization. The DNA binding domain of C is a HTH domain which is preceded by the leucine zipper motif. The C protein is one of the few examples in the bacterial proteins containing both leucine zipper and HTH domain. Most of the transcription activators either influence initial binding of RNAP or conversion of closed to open complex formation. Very few activators act at subsequent steps of promoter-polymerase interaction. Earlier studies showed high level of transcription from a mutant mom promoter, tin7. Addition of C further increased transcription from Ptin7 indicating that C may have a role beyond polymerase recruitment. Each steps of transcription initiation have been dissected using the Ptin7 and a positive control (pc) mutant of C, R105D. The results revealed multi-step transcription activation mechanism for C protein at Pmom. C recruits RNAP at Pmom and subsequently increases the productive RNAP-promoter complex and enhances promoter clearance. To further understand the C mediated transactivation mechanism, interaction between C and RNAP was assessed. C interacts with holo and core RNAP only in presence of DNA. Positive control mutants of C, F95A and R015D, were found to be compromised in RNAP interactions. These mutants were efficient in RNAP recruitment to Pmom but do not enhance promoter clearance. Trypsin cleavage protection experiment indicated that probably C protein interacts with b¢ subunit of RNAP. Interaction between C and RNAP appears to enhance the formation of productive RNAP-promoter complex leading to promoter clearance. The connection between activator-polymerase interaction and transcription activation is well documented where the recruitment of RNAP is influenced. In case of activators acting at post recruitment steps of initiation, the role of polymerase contact is poorly understood. Our study shows that activator-polymerase interaction can lead to increased promoter clearance at Pmom by overcoming abortive initiation.

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)

<p>The ability to determine nucleic acid sequences is one ofthe most important platforms for the detailed study ofbiological systems. Pyrosequencing technology is a relativelynovel DNA sequencing technique with multifaceted uniquecharacteristics, adjustable to different strategies, formatsand instrumentations. The aims of this thesis were to improvethe chemistry of the Pyrosequencing technique for increasedread-length, enhance the general sequence quality and improvethe sequencing performance for challenging templates. Improvedchemistry would enable Pyrosequencing technique to be used fornumerous applications with inherent advantages in accuracy,flexibility and parallel processing.</p><p>Pyrosequencing technology, at its advent, was restricted tosequencing short stretches of DNA. The major limiting factorwas presence of an isomer of dATPaS, a substitute for thenatural dATP, which inhibited enzyme activity in thePyrosequencing chemistry. By removing this non-functionalnucleotide, we were able to achieve DNA read-lengths of up toone hundred bases, which has been a substantial accomplishmentfor performance of different applications. Furthermore, the useof a new polymerase, called Sequenase, has enabled sequencingof homopolymeric T-regions, which are challenging for thetraditional Klenow polymerase. Sequenase has markedly madepossible sequencing of such templates with synchronizedextension.</p><p>The improved read-length and chemistry has enabledadditional applications, which were not possible previously.DNA sequencing is the gold standard method for microbial andvial typing. We have utilized Pyrosequencing technology foraccurate typing ofhuman papillomaviruses, and bacterial andfungal identification with promising results.</p><p>Furthermore, DNA sequencing technologies are not capable oftyping of a sample harboring a multitude of species/types orunspecific amplification products. We have addressed theproblem of multiple infections/variants present in a clinicalsample by a new versatile method. The multiple sequencingprimer method is suited for detection and typing of samplesharboring different clinically important types/species(multiple infections) and unspecific amplifications, whicheliminates the need for nested PCR, stringent PCR conditionsand cloning. Furthermore, the method has proved to be usefulfor samples containing subdominant types/species, and sampleswith low PCR yield, which avoids reperforming unsuccessfulPCRs. We also introduce the sequence pattern recognition whenthere is a plurality of genotypes in the sample, whichfacilitates typing of more than one target DNA in the sample.Moreover, target specific sequencing primers could be easilytailored and adapted according to the desired applications orclinical settings based on regional prevalence ofmicroorganisms and viruses.</p><p>Pyrosequencing technology has also been used forclone-checking by using preprogrammed nucleotide additionorder, EST sequencing and SNP analysis, yielding accurate andreliable results.</p><p><b>Keywords:</b>apyrase, bacterial identification, dATPaS, ESTsequencing, fungal identification, human papillomavirus (HPV),microbial and viral typing, multiple sequencing primer method,Pyrosequencing technology, Sequenase, single-strandedDNA-binding protein (SSB), SNP analysis</p>

Dissertation (Ph.D.)--University of South Florida, 2007. / Includes vita. Includes bibliographical references. Also available online.

In this thesis, a 4.7Å crystal structure of the human mitochondria DNA polymerase γ catalytic complex is reported. Though the DNA substrate-binding site is not identifiable in the structure, two conformational changes in the enzyme architecture are described: 1) rotation of the distal monomer of the accessory subunit towards the catalytic subunit, and 2) shift of the thumb motif of the polymerase domain towards the active site. Both conformational changes suggest a structure of Pol γ in the DNA-bound state and in its active site “closed” conformation. / text

No description available.

No description available.

Les dinoflagellés jouent un rôle très important dans l’écologie des océans en y réalisant une grande partie de la production primaire, en formant une association symbiotique avec les coraux et en ayant la capacité de produire des fleurs d’algues potentiellement toxiques pour les communautés côtières humaines et animales. Malgré tout, la biologie moléculaire des dinoflagellés n’a que très peu été étudiée dans les dernières années, les connaissances de processus de base comme la régulation de la transcription y étant fortement limitées. Une tentative pour élucider ce mécanisme a été réalisée chez les dinoflagellés photosynthétiques Lingulodinium polyedrum et Amphidinium carterae. Une expérience d’induction de la transcription du gène de la Peridinin chlorophyll-a binding protein, le complexe majeur de collecte de lumière, a été réalisée par une baisse de l’intensité lumineuse et a montré une faible augmentation (moins de 2 fois) du transcrit à court et long terme. Des expériences de simple-hybride et de retard sur gel (EMSA) ont été faits pour identifier de potentielles interactions protéine-ADN dans la région intergénique du gène PCP organisé en tandem. Ces essais ont été infructueux pour identifier de telles protéines. Une analyse du transcriptome de L. polyedrum a été effectuée, montrant une importante sous-représentation de domaines de liaison à l’ADN classique (comme Heat-shock factor, bZIP ou Myb) et une surreprésentation du domaine d’origine bactérienne Cold shock en comparaison avec d’autres eucaryotes unicellulaires. Ce travail suggère que les mécanismes de régulation transcriptionnelle des dinoflagellés pourraient différer substantiellement de ceux des autres eucaryotes. / Dinoflagellates are an important part of the ocean’s ecology due to their large contribution to global carbon fixation, the symbiotic association they can make with corals and by their ability to form algal blooms potentially toxic for humans and animals in coastal communities. However, the molecular biology of dinoflagellates has been poorly studied in the past. Basic knowledge, such as regulation of gene expression, is severely limited. An attempt at deciphering basic gene regulation has been undertaken in the photosynthetic dinoflagellate Lingulodinium polyedrum and Amphidinium carterae using a reduction in available light intensity to induce the expression of the peridinin chlorophyll-a binding gene encoding the major light harvesting complex protein. A small increase in transcript abundance (less than 2 fold) was found in both short and long term experiments, yet neither yeast one-hybrid assays nor electrophoretic mobility shift assays (EMSA) showed any potential protein interactions with sequence derived from the intergenic spacer of the PCP tandem gene array. Interestingly, an analysis of the recently sequenced L. polyedrum transcriptome revealed an important under-representation of classic DNA-binding domains (such has Heat-shock factor, bZIP and Myb) and an over-representation of the bacterial cold-shock DNA-binding domain. This suggested that components of the transcription regulation machinery may be at least partially different in dinoflagellates.

Malgré les progrès des traitements des cancers du sein, ceux-ci demeurent la seconde cause de mortalité par cancer au Canada. Parmi les gènes associés aux cancers du sein, le récepteur des œstrogènes ERα est exprimé dans plus de 70% des tumeurs mammaires, qui prolifèrent en réponse aux œstrogènes, faisant de lui une cible de choix. ERα est un facteur de transcription ligand-dépendant, liant des éléments de réponse PuGGTCAnnnTGACCPy. Afin d’examiner la capacité des récepteurs nucléaires à reconnaitre de nouveaux motifs ADN, des mutants aux capacités de liaison modifiées ont été générés. Parmi les quatre résidus interagissant avec l’ADN, R211 ne peut pas être modifiée sans perdre complètement la liaison du récepteur à l’ADN. Néanmoins, les mutations combinées de plusieurs acides aminés contactant les bases de l’ERE ont généré des récepteurs capables de reconnaitre de nouveaux motifs, tout en conservant des niveaux de transactivation efficaces. L’utilisation potentielle des récepteurs nucléaires comme outils de thérapie génique hormono-dépendant, repose sur la prédiction des motifs de liaison efficaces. Étant donné son importance dans la carcinogenèse mammaire, ERα est une cible cruciale des thérapies anti-néoplastiques. L’anti-œstrogène total, ICI, induit la dégradation de ERα et l’arrêt de la croissance des cellules tumorales mammaires ERα-positives. De plus, la nouvelle drogue anti-tumorale HDACi, SAHA, module la voie de signalisation des œstrogènes et possède des propriétés prometteuses en association avec d’autres traitements anti-tumoraux. En effet, le co-traitement ICI et SAHA a un impact synergique sur l’inhibition de la prolifération des cellules mammaires tumorales ERα-positives. Cette synergie repose sur la coopération des effets de ICI et SAHA pour réduire les niveaux protéiques de ERα et bloquer la progression du cycle cellulaire via la modulation de la transcription des gènes cibles des œstrogènes. En fait, les fortes doses de HDACis masquent rapidement et complètement la signalisation transcriptionnelle des œstrogènes. De plus, les gènes cibles primaires des œstrogènes, contenant des EREs, présentent la même régulation transcriptionnelle en réponse aux fortes doses de SAHA ou du co-traitement, avec des doses utilisables en clinique de ICI et SAHA. En fait, ICI mime l’impact des fortes doses de SAHA, en dégradant ERα, potentialisant ainsi la répression de la transcription ERE-dépendante par SAHA. Finalement, la synergie des effets de ICI et SAHA pourrait augmenter l’efficacité des traitements des tumeurs mammaires. / Despite the progress in breast cancer therapy, it is still the second cause of death by cancer in the western world. The estrogen receptor alpha (ER) is expressed in more than 70% of breast tumors, which then proliferate in response to E2 stimulation. ERα is a ligand-dependant transcription factor, which binds to palindromic response elements composed of PuGGTCA motifs. In order to examine the capacity of nuclear receptors to be tailored to recognize novel DNA motifs, rational design of ERα mutants with altered binding specificity were achieved. Of the four amino-acids interacting with DNA, R211 cannot be altered to another residu without a complete loss of DNA binding. However, combined mutagenesis of the amino-acids contacting the bases of the ERE generate receptors that recognized a new type of motifs with efficient transcriptional transactivation levels. In order to consider nuclear receptors as potential hormonal-dependent tools for genetic therapy, bio-informatic models predicting DNA binding are needed. Considering its importance in breast carcinogenesis, ERα is a crucial target for anti-neoplastic therapies. The full anti-estrogen ICI provokes ER degradation and ER-positive breast cancer growth arrest. Moreover, the new HDACi anti-tumoral drug, SAHA modulates the estrogen pathway and have promising properties in association with other anti-neoplastic drugs. In this context, our goal was to determine the effects of the ICI and SAHA combined treatment on breast cancer cell proliferation. The combined treatment has a synergistic impact on inhibiting ERα-positive breast cancer cell growth. It occurs through the cooperation of ICI and SAHA to reduce ER protein level, and to block the cell cycle progression through the transcriptional regulation of E2-target genes. Actually, high doses of HDACis completely and rapidly mask E2-transcriptional signaling. Moreover, primary E2-target genes show the same transcriptional regulation in presence of high doses of SAHA as with the co-treatment, using clinically feasible doses of ICI and SAHA. In fact, ICI mimics the impact of high doses of SAHA through ER degradation, and then potentiate the repressional effect of low doses of SAHA on ERE-dependant transcription. Finally, the synergy of ICI and SAHA might increase the potency of breast cancer therapy.