Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 100-109).

DNA topoisomerase I (TopoI) is an essential enzyme involved in the relief of DNA supercoiling during replication. TopoI plays important role in various DNA events, however the recognition that it is the target of anticancer drug camptothecins (CPTs) led to the rapid growth in this field. CPTs inhibit TopoI during S phase and cause double stranded DNA lesions in rapidly dividing cells. This class of drug is used extensively in oncology clinical settings worldwide. However, resistance to this type of therapy has been found in approximately 70% of the patient population. Current evidence supports that degradation of TopoI by the Ubiquitin Proteasomal Pathway (UPP), and consequent compensation by Topoisomerase II expression may be involved in imparting drug resistance, but this mechanism requires much greater understanding. Protein-protein interaction studies have indicated that, BRCA1 is the E3 ligase for TopoI ubiquitination in response to CPT. BRCA1 impaired cells fail to ubiquitinate and degrade TopoI and are sensitive to CPTs. It is important to note that triple negative breast cancer patients have impaired BRCA1 function, higher mutation rate and/or a lower expression of BRCA1. The Bharti lab has shown that TopoI associates with BRCA1. Our work attempts to elucidate the nature of the interaction between BRCA1 and TopoI in the hope of better understanding the mechanism of resistance to camptothecin therapy in TNBC.

Thesis (Ph. D.)--Rutgers University, 2008. / "Graduate Program in Medicinal Chemistry." Includes bibliographical references (p. 198-218).

Escherichia coli topoisomerase I (TopA), a regulator of global and local DNA supercoiling, is modified by Nε-Lysine acetylation. The sirtuin protein deacetylase CobB can reverse both enzymatic and non-enzymatic lysine acetylation modifications. Here, we explored the effect of lysine acetylation on E. coli topoisomerase I through analysis of TopA relaxation activity and protein expression in cell extract of wild-type and a ΔcobB mutant strains. We showed that the absence of deacetylase CobB in a ΔcobB mutant reduced intracellular TopA relaxation activity while elevating TopA expression and topA gene transcripts levels. Acetyl phosphate mediated lysine acetylation decreased the activity of purified TopA in vitro, and the interaction with purified CobB protected TopA from such inactivation. We explored the physiological significance of TopA acetylation on DNA supercoiling by two-dimensional gel analysis and on cell growth rate by growth curve analysis. We found that the absence of CobB increased negative DNA supercoiling. The slow growth phenotype of the ∆cobB mutant can be partially compensated by overexpression of recombinant TopA. In addition, the specific activity of TopA expressed from His-tagged fusion construct in the chromosome was inversely proportional to the degree of in vivo lysine acetylation during growth transition and growth arrest. Investigation of TopA relaxation mechanism using nuclease footprinting and TopA oxidative crosslinking suggested the potential association of TopA acetylation in catalysis. Mass spectrometry analysis of in vitro acetyl phosphate acetylated TopA identified abundant lysine acetylation sites. Substitution of lysine residues by site-directed mutagenesis was used to model the effect of acetylation on individual lysine residues. Our results showed that substitution of Lys-484 with alanine reduced the relaxation activity, suggesting the reduction of TopA relaxation activity by acetylation was probably in part due to acetylation on Lys-484. These findings demonstrate that E. coli topoisomerase I is modulated by lysine acetylation and the prevention of TopA inactivation from excess lysine acetylation and consequent increase in negative DNA supercoiling is an important physiological function of the sirtuin deacetylase CobB.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Acridinas sÃo molÃculas policÃclicas aromÃticas planares que possuem a capacidade de intercalar no DNA nuclear. Muitos dos seus representantes apresentam propriedades antibacterianas, antiparasitÃrias e antitumorais. O presente estudo avaliou o potencial citotÃxico de 22 novos compostos acridÃnicos em linhagens de cÃlulas tumorais humanas. Dentre esses, quatro compostos [5-(acridin-9-il-metileno)-3-(4-metil-benzil)-tiazolidina-2,4-diona, AC4; 5-(acridin-9-il-metileno)-3-(4-bromo-benzil)-tiazolidina-2,4-diona, AC7; 5-(acridin-9-il-metileno)-3-(4-cloro-benzil)-tiazolidina-2,4-diona, AC10; e 5-(acridin-9-il-metileno)-3-(4-flÃor-benzil)-tiazolidina-2,4-diona, AC23] foram ativos, especialmente em HCT-8 (cÃlon) e SF-296 (glioblastoma), com valores de CI50 variando de 2,3 a 5,3 Âg/mL. Os compostos apresentaram seletividade para cÃlulas tumorais, desde que nÃo inibiram (IC50 > 25 Âg/mL) a proliferaÃÃo de cÃlulas monocucleares de sangue perifÃrico humano (CMSPH), bem como, nÃo foram capazes de induzir dano ao DNA dessas cÃlulas. Nenhum dos compostos mostrou atividade hemolÃtica contra eritrÃcitos de camundongos (EC50 > 200 Âg/mL), o que sugere uma citotoxicidade por mecanismos mais especÃficos. A fim de determinar o mecanismo envolvido na citotoxicidade seletiva dos compostos, foi realizada uma seqÃÃncia de experimentos in vitro, usando a linhagem HCT-8 como modelo. As cÃlulas foram tratadas em diferentes concentraÃÃes dos compostos (2,5; 5 e 10 Âg/mL) por 12 e 24 horas. Todos os compostos foram capazes de reduzir a viabilidade (teste do azul de tripan) e a proliferaÃÃo (ensaio do BrdU) de cÃlulas HCT-8 apÃs o tratamento. A induÃÃo de apoptose pelos derivados acridÃnicos foi determinada por citometria de fluxo (integridade da membrana, fragmentaÃÃo do DNA internucleosomal e potencial transmembrÃnico) e por anÃlise morfolÃgica das alteraÃÃes celulares (brometo de etÃdeo/laranja de acridina e hematoxilina/eosina). A anÃlise por citometria de fluxo revelou que os compostos avaliados promoveram despolarizaÃÃo mitocondrial, o qual evidencia a ativaÃÃo da apoptose pela via intrÃnseca nas cÃlulas HCT-8. Na anÃlise do screening em 3 diferentes linhagens mutantes de Saccharomyces cerevisiae, foi observado que a linhagem Top1Δ (sem topoisomerase I) mostrou moderada resistÃncia aos compostos acridÃnicos testados na concentraÃÃo de 50 Âg/mL. AlÃm disso, as acridinas inibiram parcialmente o relaxamento do DNA por topoisomerase I, sugerindo que os compostos AC4, AC7, AC10, AC23 tem o potencial antiproliferativo, em parte, relacionado a inibiÃÃo da atividade catalÃtica desta enzima. Esses dados apontam o potencial anticÃncer dos compostos testados. / Acridines are planar aromatic polycyclic molecules that have the ability to progress in nuclear DNA. Many of its representatives have antibacterial, antiparasitic and antitumor. This study evaluated the cytotoxic potential of 22 new acridine compounds in strains of human tumor cells. Among these, four compounds [5-(acridin-9-yl-methilene)-3-(4-methyl-benzyl)-thiazolidine-2,4-dione, AC4; 5-(acridin-9-yl-methilene)-3-(4-bromine-benzyl)-thiazolidine-2,4-dione, AC7; 5-(acridin-9-yl-methilene)-3-(4-chloro-benzyl)-thiazolidine-2,4-dione, AC10; and 5-(acridin-9-yl-methilene)-3-(4-fluor-benzyl)-thiazolidine-2,4-dione, AC23] were active, especially in HCT-8 (colon) and SF-296 (glioblastoma), with IC50 values ranging from 2.3 to 5.3 mg / mL. The compounds were selective for tumor cells, provided they do not inhibit (IC50 > 25 Âg/mL) cell proliferation monocucleares human peripheral blood (CMSPH) and were not able to induce DNA damage to these cells. None of the compounds showed hemolytic activity against erythrocytes of mice (EC50 > 200 Âg/mL), suggesting a cytotoxicity by more specific mechanisms. In order to determine the mechanism involved in the selective cytotoxicity of compounds was carried out a sequence of in vitro experiments, using the line HCT-8 as a model. The cells were treated in different concentrations of compounds (2.5, 5 and 10 Âg/mL) for 12 and 24 hours. All compounds were able to reduce the viability test (trypan blue) and proliferation (BrdU assay) of HCT-8 cells after treatment. Induction of apoptosis by acridine derivatives was determined by flow cytometry (membrane integrity, DNA fragmentation and internucleosomal transmembrane potential) and morphological analysis of cellular changes (ethidium bromide/acridine orange, and hematoxylin-eosin). The analysis by flow cytometry showed that the compounds evaluated promoted mitochondrial depolarization, which shows the activation of apoptosis by intrinsic pathway in HCT-8 cells. In the analysis of screening in 3 different mutant strains of Saccharomyces cerevisiae, it was observed that the line Top1Δ (without topoisomerase I) showed resistance to acridine compounds tested at a concentration of 50 Âg/mL. In addition, the acridines partially inhibited the relaxation of DNA by topoisomerase I, suggesting that the compounds AC4, AC7, AC10, AC23 has the potential antiproliferative partly related to inhibition of catalytic activity of this enzyme. These data indicate the potential anticancer compounds tested.

Le cancer est aujourd’hui un problème de santé majeur et fait l’objet de multiples recherches. De nombreuses molécules ont été synthétisées dans l’optique de trouver des médicaments plus efficaces, plus sélectifs et surtout présentant moins d’effets secondaire. Parmi ces molécules se trouvent les lamellarines. Les lamellarines sont des composés naturels d’origine marine ayant été isolés à partir du mollusque posobranche « Lamellaria ». Actuellement, plus de 35 lamellarines ont été isolées et identifiées et certaines d’entre-elles présentent des activités biologiques intéressantes. Parmi elles certaines sont efficaces contre des cellules cancéreuses de type MDR (Multi-Drug-Resistant). Dans le cadre de la recherche de nouveaux agents cytotoxiques et d’inhibiteurs de kinase toujours plus sélectifs, la structure des lamellarines a été simplifiée par réduction du nombre de cycles et par introduction de l’indole comme coeur de la molécule. Les activités biologiques, particulièrement intéressantes, sont également rapportées dans le document. / Cancer is nowadays a major problem of public health and is the subject of many researches. Numerous molecules have been synthetized with the aim of finding more efficient and selective drugs with less secondary effects. The lamellarins can be found among these molecules. Lamellarins are a family of marine alkaloids, isolated from the prosobranch mollusk « Lamellaria ». More than 35 lamellarins have been isolated to date and only a few show interesting bioactive properties. Moreover, these compounds have been reported for their efficiency in the treatement of multi-drug resistant (MDR). In the course to design original active products, the structure of lamellarins was simplified by reduction of the number of rings and the introduction of an indol as the core of the molecule. The biological activities are reported in the document and analysed.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 103-112).

Submitted by Ramon Santana (ramon.souza@ufpe.br) on 2015-03-05T12:40:14Z No. of bitstreams: 2 Dissertação Elizabeth definitiva 2013.pdf: 2088443 bytes, checksum: e5072e4e506e5dbf48fdac9e6edd81fc (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-05T12:40:14Z (GMT). No. of bitstreams: 2 Dissertação Elizabeth definitiva 2013.pdf: 2088443 bytes, checksum: e5072e4e506e5dbf48fdac9e6edd81fc (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2013 / FACEPE (Fundação de Amparo a Ciência e Tecnologia do Estado de Pernambuco) / Muitos agentes terapêuticos essenciais no tratamento de diversas doenças atuam através da sua capacidade de interagir com o DNA. Esta interação pode muitas vezes levar à uma alteração nas propriedades estruturais e funcionais do DNA, o que influencia sobre suas funções fisiológicas e causa apoptose celular. Compostos com uma estrutura aromática policíclica, tais como acridina e seus derivados são conhecidos por interagir com o DNA e apresentar aplicações clínicas importantes, especialmente como antitumoral. Além de, atuarem através da inibição da atividade da enzima topoisomerase, bloqueando processos de replicação e transcrição do DNA, o que causa destruição das células. Dessa forma, este trabalho teve como objetivo a síntese e caracterização de derivados tiazacridínicos e imidazacridínicos para estudo da ligação ao DNA e da inibição da topoisomerase I. Todos os compostos tiveram suas estruturas elucidadas e comprovadas por RMN1H, RMN13C, IV e LC-MS. A análise da ligação ao DNA foi realiza através da espectroscopia de absorção, fluorescência e dicroísmo circular e o estudo da topoisomerase por eletroforese em gel de agarose. Os espectros de Uv-vis, fluorescência e de dicroísmo circular mostraram que os derivados interagem com o DNA tanto por ligação externa quanto por intercalação, apresentando constantes de ligação entre 1.46 – 6.01 x 104 M-1. O ensaio com a topoisomerase I evidenciou que em concentrações a partir de 200 μM, estes compostos apresentam a capacidade de inibir a enzima topoisomerase I humana. Tais resultados permitiram evidenciar um mecanismo de ligação ao DNA com as tiazacridinas e imidazacridinas, refletindo o que é visto na literatura e revelando que, estes derivados, são promissores no desenvolvimento de novos agentes análogos de acridina com potenciais sítios de ligação ao DNA.

The eubacterial genome is maintained in a negatively supercoiled state which facilitates its compaction and storage in a small cellular space. Genome supercoiling can potentially influence various DNA transaction processes such as DNA replication, transcription, recombination, chromosome segregation and gene expression. Alterations in the genome supercoiling have global impact on the gene expression and cell growth. Inside the cell, the genome supercoiling is maintained judiciously by DNA topoisomerases to optimize DNA transaction processes. These enzymes solve the problems associated with the DNA topology by cutting and rejoining the DNA. Due to their essential cellular functions and global regulatory roles, DNA topoisomerases are fascinating candidates for the study of the effect of topology perturbation on a global scale. Genus Mycobacterium includes a large number of species including the well-studied Mycobacterium smegmatis (Msm) as well as various pathogens–Mycobacterium leprae, Mycobacterium abscessus and Mycobacterium tuberculosis (Mtb), the last one being the causative agent of the deadly disease Tuberculosis (TB), which claims millions of lives worldwide annually. The organism combats various stresses and alterations in its environment during the pathogenesis and virulence. During such adaptation, various metabolic pathways and transcriptional networks are reconfigured. Considering their global regulatory role, DNA topoisomerases and genome supercoiling may have an influence on the mycobacterial survival and adaptation. Biochemical studies from our laboratory have revealed several distinctive characteristics of mycobacterial DNA gyrase and topoisomerase I. DNA gyrase has been shown to be a strong decatenase apart from its characteristic supercoiling activity. Similarly, the mycobacterial topoisomerase I exhibits several distinct features such as the ability to bind both single- as well as double-stranded DNA, site specific DNA binding and absence of Zn2+ fingers required for DNA relaxation activity in other Type I enzymes. Although, efforts have been made to understand the biochemistry and mechanism of mycobacterial topoisomerases, in vivo significance and regulatory roles remain to be explored. The present study is aimed at understanding the mechanism, in vivo functions, regulation and genome wide distribution of mycobacterial topoisomerases. Chapter 1 of the thesis provides introduction on DNA topology, genome supercoiling and DNA topoisomerases. The importance of genome supercoiling and its regulatory roles has been discussed. Further, the regulation of topoisomerase activity and the role in the virulence gene regulation is described. Finally, a brief overview of Mtb genome, disease epidemiology, and pathogenesis is presented along with the description of the work on mycobacterial topoisomerases. In Chapter 2, the studies are directed to understand the DNA relaxation mechanism of mycobacterial Type IA topoisomerase which lack Zn2+ fingers. The N-terminal domain (NTD) of the Type IA topoisomerases harbor DNA cleavage and religation activities, but the carboxyl terminal domain (CTD) is highly diverse. Most of these enzymes contain a varied number of Zn2+ finger motifs in the CTD. The Zn2+ finger motifs were found to be essential in Escherichia coli TopoI but dispensable in the Thermotoga maritima enzyme. Although, the CTD of mycobacterial TopoI lacks Zn2+ fingers, it is indispensable for the DNA relaxation activity of the enzyme. The divergent CTD harbors three stretches of basic amino acids needed for the strand passage step of the reaction as demonstrated by a new assay. It is elucidated that the basic amino acids constitute an independent DNA-binding site apart from the NTD and assist the simultaneous binding of two molecules of DNA to the enzyme, as required during the strand passage step of the catalysis. It is hypothesized that the loss of Zn2+ fingers from the mycobacterial TopoI could be associated with Zn2+ export and homeostasis. In Chapter 3, the studies have been carried out to understand the regulation of mycobacterial TopoI. Identification of Transcription Start Site (TSS) suggested the presence of multiple promoters which were found to be sensitive to genome supercoiling. The promoter activity was found to be specific to mycobacteria as the promoter(s) did not show activity in E. coli. Analysis of the putative promoter elements suggested the non-optimal spacing of the putative -35 and -10 promoter elements indicating the involvement of supercoiling for the optimal alignment during the transcription. Moreover, upon genome relaxation, the occupancy of RNA polymerase was decreased on the promoter region of topoI gene implicating the role of DNA topology in the Supercoiling Sensitive Transcription (SST) of TopoI gene from mycobacteria. The involvement of intrinsic promoter elements in such regulation has been proposed. In Chapter 4, the importance of TopoI for the Mtb growth and survival has been validated. Mtb contains only one Type IA topoisomerase (Rv3646c), a sole DNA relaxase in the cell, and hence a candidate drug target. To validate the essentiality of Mtb topoisomerase I for bacterial growth and survival, conditionally regulated strain of topoI in Mtb was generated. The conditional knockdown mutant exhibited delayed growth on agar plate and in liquid culture the growth was drastically impaired when TopoI expression was suppressed. Additionally, novobiocin and isoniazid showed enhanced inhibitory potential against the conditional mutant. Analysis of the nucleoid revealed its altered architecture upon TopoI depletion. These studies establish the essentiality of TopoI for the Mtb growth and open up new avenues for targeting the enzyme. In Chapter 5, the influence of perturbation of TopoI activity on the Msm growth and physiology has been studied. Notably, Msm contains an additional DNA relaxation enzyme– an atypical Type II topoisomerase TopoNM. The TopoI depleted strain exhibited slow growth and drastic change in phenotypic characters. Moreover, the genome architecture was disturbed upon depletion of TopoI. Further, the proteomic and transcript analysis indicated the altered expression of the genes involved in central metabolic pathways and core DNA transaction processes in the mutant. The study suggests the importance of TopoI in the maintenance of cellular phenotype and growth characteristics of fast growing mycobacteria having additional topoisomerases. In Chapter 6, the ChIP-Seq method is used to decipher the genome wide distribution of the DNA gyrase, topoisomerase I (TopoI) and RNA polymerase (RNAP). Analysis of the ChIP-Seq data revealed the genome wide distribution of topoisomerases along with RNAP. Importantly, the signals of topoisomerases and RNAP was found to be co-localized on the genome suggesting their functional association in the twin supercoiled domain model, originally proposed by J. C. Wang. Closer inspection of the occupancy profile of topoisomerases and RNAP on transcription units (TUs) revealed their co-existence validating the topoisomerases occupancy within the twin supercoiled domains. On the genomic scale, the distribution of topoisomerases was found to be more at the ori domains compared to the ter domain which appeared to be an attribute of higher torsional stress at ori. The reappearance of gyrase binding at the ter domain (and the lack of it in the ter domain of E. coli) suggests a role for Mtb gyrase in the decatenation of the daughter chromosomes at the end of replication. The eubacterial genome is maintained in a negatively supercoiled state which facilitates its compaction and storage in a small cellular space. Genome supercoiling can potentially influence various DNA transaction processes such as DNA replication, transcription, recombination, chromosome segregation and gene expression. Alterations in the genome supercoiling have global impact on the gene expression and cell growth. Inside the cell, the genome supercoiling is maintained judiciously by DNA topoisomerases to optimize DNA transaction processes. These enzymes solve the problems associated with the DNA topology by cutting and rejoining the DNA. Due to their essential cellular functions and global regulatory roles, DNA topoisomerases are fascinating candidates for the study of the effect of topology perturbation on a global scale. Genus Mycobacterium includes a large number of species including the well-studied Mycobacterium smegmatis (Msm) as well as various pathogens–Mycobacterium leprae, Mycobacterium abscessus and Mycobacterium tuberculosis (Mtb), the last one being the causative agent of the deadly disease Tuberculosis (TB), which claims millions of lives worldwide annually. The organism combats various stresses and alterations in its environment during the pathogenesis and virulence. During such adaptation, various metabolic pathways and transcriptional networks are reconfigured. Considering their global regulatory role, DNA topoisomerases and genome supercoiling may have an influence on the mycobacterial survival and adaptation. Biochemical studies from our laboratory have revealed several distinctive characteristics of mycobacterial DNA gyrase and topoisomerase I. DNA gyrase has been shown to be a strong decatenase apart from its characteristic supercoiling activity. Similarly, the mycobacterial topoisomerase I exhibits several distinct features such as the ability to bind both single- as well as double-stranded DNA, site specific DNA binding and absence of Zn2+ fingers required for DNA relaxation activity in other Type I enzymes. Although, efforts have been made to understand the biochemistry and mechanism of mycobacterial topoisomerases, in vivo significance and regulatory roles remain to be explored. The present study is aimed at understanding the mechanism, in vivo functions, regulation and genome wide distribution of mycobacterial topoisomerases. Chapter 1 of the thesis provides introduction on DNA topology, genome supercoiling and DNA topoisomerases. The importance of genome supercoiling and its regulatory roles has been discussed. Further, the regulation of topoisomerase activity and the role in the virulence gene regulation is described. Finally, a brief overview of Mtb genome, disease epidemiology, and pathogenesis is presented along with the description of the work on mycobacterial topoisomerases. In Chapter 2, the studies are directed to understand the DNA relaxation mechanism of mycobacterial Type IA topoisomerase which lack Zn2+ fingers. The N-terminal domain (NTD) of the Type IA topoisomerases harbor DNA cleavage and religation activities, but the carboxyl terminal domain (CTD) is highly diverse. Most of these enzymes contain a varied number of Zn2+ finger motifs in the CTD. The Zn2+ finger motifs were found to be essential in Escherichia coli TopoI but dispensable in the Thermotoga maritima enzyme. Although, the CTD of mycobacterial TopoI lacks Zn2+ fingers, it is indispensable for the DNA relaxation activity of the enzyme. The divergent CTD harbors three stretches of basic amino acids needed for the strand passage step of the reaction as demonstrated by a new assay. It is elucidated that the basic amino acids constitute an independent DNA-binding site apart from the NTD and assist the simultaneous binding of two molecules of DNA to the enzyme, as required during the strand passage step of the catalysis. It is hypothesized that the loss of Zn2+ fingers from the mycobacterial TopoI could be associated with Zn2+ export and homeostasis. In Chapter 3, the studies have been carried out to understand the regulation of mycobacterial TopoI. Identification of Transcription Start Site (TSS) suggested the presence of multiple promoters which were found to be sensitive to genome supercoiling. The promoter activity was found to be specific to mycobacteria as the promoter(s) did not show activity in E. coli. Analysis of the putative promoter elements suggested the non-optimal spacing of the putative -35 and -10 promoter elements indicating the involvement of supercoiling for the optimal alignment during the transcription. Moreover, upon genome relaxation, the occupancy of RNA polymerase was decreased on the promoter region of topoI gene implicating the role of DNA topology in the Supercoiling Sensitive Transcription (SST) of TopoI gene from mycobacteria. The involvement of intrinsic promoter elements in such regulation has been proposed. In Chapter 4, the importance of TopoI for the Mtb growth and survival has been validated. Mtb contains only one Type IA topoisomerase (Rv3646c), a sole DNA relaxase in the cell, and hence a candidate drug target. To validate the essentiality of Mtb topoisomerase I for bacterial growth and survival, conditionally regulated strain of topoI in Mtb was generated. The conditional knockdown mutant exhibited delayed growth on agar plate and in liquid culture the growth was drastically impaired when TopoI expression was suppressed. Additionally, novobiocin and isoniazid showed enhanced inhibitory potential against the conditional mutant. Analysis of the nucleoid revealed its altered architecture upon TopoI depletion. These studies establish the essentiality of TopoI for the Mtb growth and open up new avenues for targeting the enzyme. In Chapter 5, the influence of perturbation of TopoI activity on the Msm growth and physiology has been studied. Notably, Msm contains an additional DNA relaxation enzyme– an atypical Type II topoisomerase TopoNM. The TopoI depleted strain exhibited slow growth and drastic change in phenotypic characters. Moreover, the genome architecture was disturbed upon depletion of TopoI. Further, the proteomic and transcript analysis indicated the altered expression of the genes involved in central metabolic pathways and core DNA transaction processes in the mutant. The study suggests the importance of TopoI in the maintenance of cellular phenotype and growth characteristics of fast growing mycobacteria having additional topoisomerases. In Chapter 6, the ChIP-Seq method is used to decipher the genome wide distribution of the DNA gyrase, topoisomerase I (TopoI) and RNA polymerase (RNAP). Analysis of the ChIP-Seq data revealed the genome wide distribution of topoisomerases along with RNAP. Importantly, the signals of topoisomerases and RNAP was found to be co-localized on the genome suggesting their functional association in the twin supercoiled domain model, originally proposed by J. C. Wang. Closer inspection of the occupancy profile of topoisomerases and RNAP on transcription units (TUs) revealed their co-existence validating the topoisomerases occupancy within the twin supercoiled domains. On the genomic scale, the distribution of topoisomerases was found to be more at the ori domains compared to the ter domain which appeared to be an attribute of higher torsional stress at ori. The reappearance of gyrase binding at the ter domain (and the lack of it in the ter domain of E. coli) suggests a role for Mtb gyrase in the decatenation of the daughter chromosomes at the end of replication.

No description available.