Le surenroulement de l’ADN est important pour tous les processus cellulaires qui requièrent la séparation des brins de l’ADN. Il est régulé par l’activité enzymatique des topoisomérases. La gyrase (gyrA et gyrB) utilise l’ATP pour introduire des supertours négatifs dans l’ADN, alors que la topoisomérase I (topA) et la topoisomérase IV (parC et parE) les éliminent. Les cellules déficientes pour la topoisomérase I sont viables si elles ont des mutations compensatoires dans un des gènes codant pour une sous-unité de la gyrase. Ces mutations réduisent le niveau de surenroulement négatif du chromosome et permettent la croissance bactérienne. Une de ces mutations engendre la production d'une gyrase thermosensible. L’activité de surenroulement de la gyrase en absence de la topoisomérase I cause l’accumulation d’ADN hyper-surenroulé négativement à cause de la formation de R-loops. La surproduction de la RNase HI (rnhA), une enzyme qui dégrade l’ARN des R-loops, permet de prévenir l’accumulation d’un excès de surenroulement négatif. En absence de RNase HI, des R-loops sont aussi formés et peuvent être utilisés pour déclencher la réplication de l’ADN indépendamment du système normal oriC/DnaA, un phénomène connu sous le nom de « constitutive stable DNA replication » (cSDR). Pour mieux comprendre le lien entre la formation de R-loops et l’excès de surenroulement négatif, nous avons construit un mutant conditionnel topA rnhA gyrB(Ts) avec l’expression inductible de la RNase HI à partir d’un plasmide. Nous avons trouvé que l’ADN des cellules de ce mutant était excessivement relâché au lieu d'être hypersurenroulé négativement en conditions de pénurie de RNase HI. La relaxation de l’ADN a été montrée comme étant indépendante de l'activité de la topoisomérase IV. Les cellules du triple mutant topA rnhA gyrB(Ts) forment de très longs filaments remplis d’ADN, montrant ainsi un défaut de ségrégation des chromosomes. La surproduction de la topoisomérase III (topB), une enzyme qui peut effectuer la décaténation de l’ADN, a corrigé les problèmes de ségrégation sans toutefois restaurer le niveau de surenroulement de l’ADN. Nous avons constaté que des extraits protéiques du mutant topA rnhA gyrB(Ts) pouvaient inhiber l’activité de surenroulement négatif de la gyrase dans des extraits d’une souche sauvage, suggérant ainsi que la pénurie de RNase HI avait déclenché une réponse cellulaire d’inhibition de cette activité de la gyrase. De plus, des expériences in vivo et in vitro ont montré qu’en absence de RNase HI, l’activité ATP-dépendante de surenroulement négatif de la gyrase était inhibée, alors que l’activité ATP-indépendante de cette enzyme demeurait intacte. Des suppresseurs extragéniques du défaut de croissance du triple mutant topA rnhA gyrB(Ts) qui corrigent également les problèmes de surenroulement et de ségrégation des chromosomes ont pour la plupart été cartographiés dans des gènes impliqués dans la réplication de l’ADN, le métabolisme des R-loops, ou la formation de fimbriae. La deuxième partie de ce projet avait pour but de comprendre les rôles des topoisomérases de type IA (topoisomérase I et topoisomérase III) dans la ségrégation et la stabilité du génome de Escherichia coli. Pour étudier ces rôles, nous avons utilisé des approches de génétique combinées avec la cytométrie en flux, l’analyse de type Western blot et la microscopie. Nous avons constaté que le phénotype Par- et les défauts de ségrégation des chromosomes d’un mutant gyrB(Ts) avaient été corrigés en inactivant topA, mais uniquement en présence du gène topB. En outre, nous avons démontré que la surproduction de la topoisomérase III pouvait corriger le phénotype Par- du mutant gyrB(Ts) sans toutefois corriger les défauts de croissance de ce dernier. La surproduction de topoisomérase IV, enzyme responsable de la décaténation des chromosomes chez E. coli, ne pouvait pas remplacer la topoisomérase III. Nos résultats suggèrent que les topoisomérases de type IA jouent un rôle important dans la ségrégation des chromosomes lorsque la gyrase est inefficace. Pour étudier le rôle des topoisomérases de type IA dans la stabilité du génome, la troisième partie du projet, nous avons utilisé des approches génétiques combinées avec des tests de « spot » et la microscopie. Nous avons constaté que les cellules déficientes en topoisomérase I avaient des défauts de ségrégation de chromosomes et de croissance liés à un excès de surenroulement négatif, et que ces défauts pouvaient être corrigés en inactivant recQ, recA ou par la surproduction de la topoisomérase III. Le suppresseur extragénique oriC15::aph isolé dans la première partie du projet pouvait également corriger ces problèmes. Les cellules déficientes en topoisomérases de type IA formaient des très longs filaments remplis d’ADN d’apparence diffuse et réparti inégalement dans la cellule. Ces phénotypes pouvaient être partiellement corrigés par la surproduction de la RNase HI ou en inactivant recA, ou encore par des suppresseurs isolés dans la première partie du projet et impliques dans le cSDR (dnaT18::aph et rne59::aph). Donc, dans E. coli, les topoisomérases de type IA jouent un rôle dans la stabilité du génome en inhibant la réplication inappropriée à partir de oriC et de R-loops, et en empêchant les défauts de ségrégation liés à la recombinaison RecA-dépendante, par leur action avec RecQ. Les travaux rapportés ici révèlent que la réplication inappropriée et dérégulée est une source majeure de l’instabilité génomique. Empêcher la réplication inappropriée permet la ségrégation des chromosomes et le maintien d’un génome stable. La RNase HI et les topoisomérases de type IA jouent un rôle majeur dans la prévention de la réplication inappropriée. La RNase HI réalise cette tâche en modulant l’activité de surenroulement ATP-dependante de la gyrase, et en empêchant la réplication à partir des R-loops. Les topoisomérases de type IA assurent le maintien de la stabilité du génome en empêchant la réplication inappropriée à partir de oriC et des R-loops et en agissant avec RecQ pour résoudre des intermédiaires de recombinaison RecA-dépendants afin de permettre la ségrégation des chromosomes. / DNA supercoiling is important for all cellular processes that require strand separation and is regulated by the opposing enzymatic effects of DNA topoisomerases. Gyrase uses ATP to introduce negative supercoils while topoisomerase I (topA) and topoisomerase IV relax negative supercoils. Cells lacking topoisomerase I are only viable if they have compensatory mutations in gyrase genes that reduce the negative supercoiling level of the chromosome to allow bacterial growth. One such mutation leads to the production of a thermosensitive gyrase (gyrB(Ts)). Gyrase driven supercoiling during transcription in the absence of topoisomerase I causes the accumulation of hypernegatively supercoiled plasmid DNAs due to the formation of R-loops. Overproducing RNase HI (rnhA), an enzyme that degrades the RNA moiety of R-loops, prevents the accumulation of hypernegative supercoils. In the absence of RNase HI alone, R-loops are equally formed and can be used to prime DNA replication independently of oriC/DnaA, a phenomenon known as constitutive stable DNA replication (cSDR). To better understand the link between R-loop formation and hypernegative supercoiling, we constructed a conditional topA rnhA gyrB(Ts) mutant with RNase HI being conditionally expressed from a plasmid borne gene. We found that the DNA of topA rnhA gyrB(Ts) cells was extensively relaxed instead of being hypernegatively supercoiled following the depletion of RNase HI. Relaxation was found to be unrelated to the activity of topoisomerase IV. Cells of topA rnhA gyrB(Ts) formed long filaments full of DNA, consistent with segregation defect. Overproducing topoisomerase III (topB), an enzyme that can perform decatenation, corrected the segregation problems without restoring supercoiling. We found that extracts of topA rnhA gyrB(Ts) cells inhibited gyrase supercoiling activity of wild type cells extracts in vitro, suggesting that the depletion of RNase HI triggered a cell response that inhibited the supercoiling activity of gyrase. Gyrase supercoiling assays in vivo as well as in crude cell extracts revealed that the ATP dependent supercoiling reaction of gyrase was inhibited while the ATP independent relaxation reaction was unaffected. Genetic suppressors of a triple topA rnhA gyrB(Ts) strain that restored supercoiling and corrected the chromosome segregation defects mostly mapped to genes that affected DNA replication, R-loop metabolism and fimbriae formation. The second part of this project aimed at understanding the roles of type IA DNA topoisomerases (topoisomerase I and topoisomerase III) in chromosome segregation and genome maintenance in E. coli. To investigate the role of type IA DNA topoisomerases in chromosome segregation we employed genetic approaches combined with flow cytometry, Western blot analysis and microscopy (for the examination of cell morphology). We found that the Par- phenotypes (formation of large unsegregated nucleoid in midcell) and chromosome segregation defects of a gyrB(Ts) mutant at the nonpermissive temperature were corrected by deleting topA only in the presence of topB. Moreover, overproducing topoisomerase III was shown to correct the Par- phenotype without correcting the growth defect, but overproducing topoisomerase IV, the major cellular decatenase, failed to correct the defects. Our results suggest that type IA topoisomerases play a role in chromosome segregation when gyrase is inefficient. To investigate the role of type IA DNA topoisomerases in genome maintenance, in the third part of the project, we employed genetic approaches combined with suppressor screens, spot assays and microscopy. We found that cells lacking topoisomerase I suffered from supercoiling-dependent growth defects and chromosome segregation defects that could be corrected by deleting recQ, recA or overproducing topoisomerase III and by an oriC15::aph suppressor mutation isolated in the first part of the project. Cells lacking both type 1A topoisomerases formed very long filaments packed with diffuse and unsegregated DNA. Such phenotypes could be partially corrected by overproducing RNase HI or deleting recA, or by suppressor mutations isolated in the first part of the project, that affected cSDR (dnaT18::aph and rne59::aph). Thus, in E. coli, type IA DNA topoisomerases play a role in genome maintenance by inhibiting inappropriate replication from oriC and R-loops and by preventing RecA-dependent chromosome segregation defect through their action with RecQ. The work reported here reveals that inappropriate and unregulated replication is a major source of genome instability. Preventing such replication will ensures proper chromosome segregation leading to a stable genome. RNase HI and type IA DNA topoisomerases play a leading role in preventing unregulated replication. RNase HI achieves this role by modulating ATP dependent gyrase activity and by preventing replication from R-loops (cSDR). Type IA DNA topoisomerases ensure the maintenance of a stable genome by preventing inappropriate replication from oriC and R-loops and by acting with RecQ to prevent RecA dependent-chromosome segregation defects.

This thesis is concerned with synthesis, structure and biochemical analysis of chemically modified oligonucleotides with potential therapeutic applications. The three types of chemical modifications described here are: (a) A North-East locked 1',2'-azetidine nucleoside (b) A North locked 2',4'-cyanomethylene bridged nucleoside and (c) A 2',4'-aza-ENA-T nucleoside. The synthesis of the 1',2'-azetidine fused nucleosides was described using two different approaches. A highly strained 2',4'-cyanomethylene locked nucleoside was synthesized but could not be converted to the phosphoramidite derivative due to instability during derivatization. The key cyclization step in the aza-ENA-T nucleoside synthesis gave rise to two separable diastereomers due to chirality at the exocyclic nitrogen. Conversion of diastereomer 55 to 56 occurred with a large free energy of activation (ΔG‡ = 23.4 kcal mol-1 at 298 K in pyridine-d5). Of the two isomers the equatorial NH product was more stable than the axial one due to reduced 1,3 diaxial interactions. As a result, all NH axial product was converted to the equatorial isomer during subsequent steps in the synthesis. NMR and ab initio experiments confirmed the North-East structure of the 1',2'-azetidine locked nucleoside and North conformation of aza-ENA-T locked nucleosides with a chair conformation of the piperidine ring. The amino modified nucleosides were incorporated into different positions of a 15mer oligonucleotide. The azetidine modified AONs did not form stable duplexes with complementary RNA (ΔTm ~-1 to -4 °C), but they performed better than previously synthesized isosequential 1',2'-oxetane modified oligonucleotides. The 2',4'-aza-ENA-T modified oligonucleotide, on the other hand, showed excellent target affinity with complementary RNA (ΔTm ~+4 °C). The azetidine and aza-ENA-T modified oligonucleotides showed significant stability in the presence of human serum and snake venom phosphodiesterase (3'-exonuclease) as compared to the unmodified native sequence. The singly modified 15mer oligonucleotides were also subjected to RNase H promoted digestion in order to evaluate their potential as effective antisense agents. The effective enzyme activity (kcat/Km) was found to be lower in the modified AONs due to reduced enzyme-substrate binding. However, the catalytic activity of RNase H with these modified-AON:RNA duplexes were higher than observed with the native duplex.

This thesis is based on six original research publications describing synthesis, structure and physicochemical and biochemical analysis of chemically modified oligonucleotides (ONs) in terms of their potential diagnostic and therapeutic applications. Synthesis of two types of bicyclic conformationally constrained nucleosides, North-East locked 1',2'-azetidine and North locked 2',4'-aza-ENA, is described. Study of the molecular structures and dynamics of bicyclic nucleosides showed that depending upon the type of fused system they fall into two distinct categories with their respective internal dynamics and type of sugar conformation. The physicochemical properties of the nucleobases in the conformationally constrained nucleosides found to be depended on the site and ring-size of the fused system. The incorporation of azetidine modified nucleotide units into 15mer ONs lowered the affinity toward the complementary RNA. However, they performed better than previously reported isosequential 1',2'-oxetane modified analogues. Whereas aza-ENA-T modification incorporated into ONs significantly enhanced affinity to the complementary RNA. To evaluate the antisense potential of azetidine-T and aza-ENA-T modified ONs, they were subjected to RNase H promoted cleavage as well as tested towards nucleolytic degradation. Kinetic experiments showed that modified ONs recruit RNase H, however with lower enzyme efficiency due to decreased enzyme-substrate binding affinity, but with enhanced turnover number. Both, azetidine-T and aza-ENA-T modified ONs demonstrated improved 3'-exonuclease stability in the presence of snake venom phosphodiesterase and human serum compared to the unmodified sequence. Oligodeoxynucleotides (ODNs) containing pyrene-functionalized azetidine-T (Aze-pyr X) and aza-ENA-T (Aza-ENA-pyr Y) modifications showed different fluorescence properties. The X modified ODNs hybridized to the complementary DNA and RNA showed variable increase in the fluorescence intensity depending upon the nearest-neighbor at the 3'-end to X modification (dA > dG > dT > dC) with high fluorescence quantum yield. However, the Y modified ODNs showed a sensible enhancement of the fluorescence intensity only with complementary DNA. Also, the X modified ODN showed decrease (~37-fold) in the fluorescence intensity upon duplex formation with RNA containing a G nucleobase mismatch opposite to the modification site, whereas a ~3-fold increase was observed for the Y modified probe.

Les topoisomérases I (topA) et III (topB) sont les deux topoisomérases (topos) de type IA d’Escherichia coli. La fonction principale de la topo I est la relaxation de l’excès de surenroulement négatif, tandis que peu d’information est disponible sur le rôle de la topo III. Les cellules pour lesquelles les deux topoisomérases de type IA sont manquantes souffrent d’une croissance difficile ainsi que de défauts de ségrégation sévères. Nous démontrons que ces problèmes sont majoritairement attribuables à des mutations dans la gyrase qui empêchent l’accumulation d’excès de surenroulement négatif chez les mutants sans topA. L’augmentation de l’activité de la gyrase réalisée par le remplacement de l’allèle gyrB(Ts) par le gène de type sauvage ou par l’exposition des souches gyrB(Ts) à une température permissive, permet la correction significative de la croissance et de la ségrégation des cellules topos de type IA. Nous démontrons également que les mutants topB sont hypersensibles à l’inhibition de la gyrase par la novobiocine. La réplication non-régulée en l’absence de topA et de rnhA (RNase HI) augmente la nécessité de l’activité de la topoisomérase III. De plus, en l’absence de topA et de rnhA, la surproduction de la topoisomérase III permet de réduire la dégradation importante d’ADN qui est observée en l’absence de recA (RecA). Nous proposons un rôle pour la topoisomérase III dans la ségrégation des chromosomes lorsque l’activité de la gyrase n’est pas optimale, par la réduction des collisions fourches de réplication s’observant particulièrement en l’absence de la topo I et de la RNase HI. / E. coli possesses two type IA topoisomerases (topos), namely topo I (topA) and topo III (topB). The major function of topo I is the relaxation of excess negative supercoiling. Much less is known about the function of topo III. Cells lacking both type IA topos suffer from severe chromosome segregation and growth defects. We show that these defects are mostly related to the presence of gyrase mutations that prevent excess negative supercoiling in topA null mutants. Indeed, increasing gyrase activity by spontaneous mutations, by substituting a gyrB(Ts) allele for a wild-type one or by exposing cells carrying the gyrB(Ts) allele to permissive temperatures, significantly corrected the growth and segregation defects of cells lacking type IA topo activity. We also found that topB mutants are hypersensitive to novobiocin due to gyrase inhibition. Our data also suggest that unregulated replication occurring in the absence of topA and rnhA (RNase HI) exacerbates the need for topo III activity. Moreover, when topA and rnhA were absent, we found that topo III overproduction reduced the extensive DNA degradation that took place in the absence of recA (RecA). All together, our results lead us to propose a role for topo III in chromosome segregation when gyrase activity is suboptimal, thus reducing replication forks collapse, especially when replication is unregulated due to the absence of topo I and RNase HI.

Le surenroulement de l’ADN est important pour tous les processus cellulaires qui requièrent la séparation des brins de l’ADN. Il est régulé par l’activité enzymatique des topoisomérases. La gyrase (gyrA et gyrB) utilise l’ATP pour introduire des supertours négatifs dans l’ADN, alors que la topoisomérase I (topA) et la topoisomérase IV (parC et parE) les éliminent. Les cellules déficientes pour la topoisomérase I sont viables si elles ont des mutations compensatoires dans un des gènes codant pour une sous-unité de la gyrase. Ces mutations réduisent le niveau de surenroulement négatif du chromosome et permettent la croissance bactérienne. Une de ces mutations engendre la production d'une gyrase thermosensible. L’activité de surenroulement de la gyrase en absence de la topoisomérase I cause l’accumulation d’ADN hyper-surenroulé négativement à cause de la formation de R-loops. La surproduction de la RNase HI (rnhA), une enzyme qui dégrade l’ARN des R-loops, permet de prévenir l’accumulation d’un excès de surenroulement négatif. En absence de RNase HI, des R-loops sont aussi formés et peuvent être utilisés pour déclencher la réplication de l’ADN indépendamment du système normal oriC/DnaA, un phénomène connu sous le nom de « constitutive stable DNA replication » (cSDR). Pour mieux comprendre le lien entre la formation de R-loops et l’excès de surenroulement négatif, nous avons construit un mutant conditionnel topA rnhA gyrB(Ts) avec l’expression inductible de la RNase HI à partir d’un plasmide. Nous avons trouvé que l’ADN des cellules de ce mutant était excessivement relâché au lieu d'être hypersurenroulé négativement en conditions de pénurie de RNase HI. La relaxation de l’ADN a été montrée comme étant indépendante de l'activité de la topoisomérase IV. Les cellules du triple mutant topA rnhA gyrB(Ts) forment de très longs filaments remplis d’ADN, montrant ainsi un défaut de ségrégation des chromosomes. La surproduction de la topoisomérase III (topB), une enzyme qui peut effectuer la décaténation de l’ADN, a corrigé les problèmes de ségrégation sans toutefois restaurer le niveau de surenroulement de l’ADN. Nous avons constaté que des extraits protéiques du mutant topA rnhA gyrB(Ts) pouvaient inhiber l’activité de surenroulement négatif de la gyrase dans des extraits d’une souche sauvage, suggérant ainsi que la pénurie de RNase HI avait déclenché une réponse cellulaire d’inhibition de cette activité de la gyrase. De plus, des expériences in vivo et in vitro ont montré qu’en absence de RNase HI, l’activité ATP-dépendante de surenroulement négatif de la gyrase était inhibée, alors que l’activité ATP-indépendante de cette enzyme demeurait intacte. Des suppresseurs extragéniques du défaut de croissance du triple mutant topA rnhA gyrB(Ts) qui corrigent également les problèmes de surenroulement et de ségrégation des chromosomes ont pour la plupart été cartographiés dans des gènes impliqués dans la réplication de l’ADN, le métabolisme des R-loops, ou la formation de fimbriae. La deuxième partie de ce projet avait pour but de comprendre les rôles des topoisomérases de type IA (topoisomérase I et topoisomérase III) dans la ségrégation et la stabilité du génome de Escherichia coli. Pour étudier ces rôles, nous avons utilisé des approches de génétique combinées avec la cytométrie en flux, l’analyse de type Western blot et la microscopie. Nous avons constaté que le phénotype Par- et les défauts de ségrégation des chromosomes d’un mutant gyrB(Ts) avaient été corrigés en inactivant topA, mais uniquement en présence du gène topB. En outre, nous avons démontré que la surproduction de la topoisomérase III pouvait corriger le phénotype Par- du mutant gyrB(Ts) sans toutefois corriger les défauts de croissance de ce dernier. La surproduction de topoisomérase IV, enzyme responsable de la décaténation des chromosomes chez E. coli, ne pouvait pas remplacer la topoisomérase III. Nos résultats suggèrent que les topoisomérases de type IA jouent un rôle important dans la ségrégation des chromosomes lorsque la gyrase est inefficace. Pour étudier le rôle des topoisomérases de type IA dans la stabilité du génome, la troisième partie du projet, nous avons utilisé des approches génétiques combinées avec des tests de « spot » et la microscopie. Nous avons constaté que les cellules déficientes en topoisomérase I avaient des défauts de ségrégation de chromosomes et de croissance liés à un excès de surenroulement négatif, et que ces défauts pouvaient être corrigés en inactivant recQ, recA ou par la surproduction de la topoisomérase III. Le suppresseur extragénique oriC15::aph isolé dans la première partie du projet pouvait également corriger ces problèmes. Les cellules déficientes en topoisomérases de type IA formaient des très longs filaments remplis d’ADN d’apparence diffuse et réparti inégalement dans la cellule. Ces phénotypes pouvaient être partiellement corrigés par la surproduction de la RNase HI ou en inactivant recA, ou encore par des suppresseurs isolés dans la première partie du projet et impliques dans le cSDR (dnaT18::aph et rne59::aph). Donc, dans E. coli, les topoisomérases de type IA jouent un rôle dans la stabilité du génome en inhibant la réplication inappropriée à partir de oriC et de R-loops, et en empêchant les défauts de ségrégation liés à la recombinaison RecA-dépendante, par leur action avec RecQ. Les travaux rapportés ici révèlent que la réplication inappropriée et dérégulée est une source majeure de l’instabilité génomique. Empêcher la réplication inappropriée permet la ségrégation des chromosomes et le maintien d’un génome stable. La RNase HI et les topoisomérases de type IA jouent un rôle majeur dans la prévention de la réplication inappropriée. La RNase HI réalise cette tâche en modulant l’activité de surenroulement ATP-dependante de la gyrase, et en empêchant la réplication à partir des R-loops. Les topoisomérases de type IA assurent le maintien de la stabilité du génome en empêchant la réplication inappropriée à partir de oriC et des R-loops et en agissant avec RecQ pour résoudre des intermédiaires de recombinaison RecA-dépendants afin de permettre la ségrégation des chromosomes. / DNA supercoiling is important for all cellular processes that require strand separation and is regulated by the opposing enzymatic effects of DNA topoisomerases. Gyrase uses ATP to introduce negative supercoils while topoisomerase I (topA) and topoisomerase IV relax negative supercoils. Cells lacking topoisomerase I are only viable if they have compensatory mutations in gyrase genes that reduce the negative supercoiling level of the chromosome to allow bacterial growth. One such mutation leads to the production of a thermosensitive gyrase (gyrB(Ts)). Gyrase driven supercoiling during transcription in the absence of topoisomerase I causes the accumulation of hypernegatively supercoiled plasmid DNAs due to the formation of R-loops. Overproducing RNase HI (rnhA), an enzyme that degrades the RNA moiety of R-loops, prevents the accumulation of hypernegative supercoils. In the absence of RNase HI alone, R-loops are equally formed and can be used to prime DNA replication independently of oriC/DnaA, a phenomenon known as constitutive stable DNA replication (cSDR). To better understand the link between R-loop formation and hypernegative supercoiling, we constructed a conditional topA rnhA gyrB(Ts) mutant with RNase HI being conditionally expressed from a plasmid borne gene. We found that the DNA of topA rnhA gyrB(Ts) cells was extensively relaxed instead of being hypernegatively supercoiled following the depletion of RNase HI. Relaxation was found to be unrelated to the activity of topoisomerase IV. Cells of topA rnhA gyrB(Ts) formed long filaments full of DNA, consistent with segregation defect. Overproducing topoisomerase III (topB), an enzyme that can perform decatenation, corrected the segregation problems without restoring supercoiling. We found that extracts of topA rnhA gyrB(Ts) cells inhibited gyrase supercoiling activity of wild type cells extracts in vitro, suggesting that the depletion of RNase HI triggered a cell response that inhibited the supercoiling activity of gyrase. Gyrase supercoiling assays in vivo as well as in crude cell extracts revealed that the ATP dependent supercoiling reaction of gyrase was inhibited while the ATP independent relaxation reaction was unaffected. Genetic suppressors of a triple topA rnhA gyrB(Ts) strain that restored supercoiling and corrected the chromosome segregation defects mostly mapped to genes that affected DNA replication, R-loop metabolism and fimbriae formation. The second part of this project aimed at understanding the roles of type IA DNA topoisomerases (topoisomerase I and topoisomerase III) in chromosome segregation and genome maintenance in E. coli. To investigate the role of type IA DNA topoisomerases in chromosome segregation we employed genetic approaches combined with flow cytometry, Western blot analysis and microscopy (for the examination of cell morphology). We found that the Par- phenotypes (formation of large unsegregated nucleoid in midcell) and chromosome segregation defects of a gyrB(Ts) mutant at the nonpermissive temperature were corrected by deleting topA only in the presence of topB. Moreover, overproducing topoisomerase III was shown to correct the Par- phenotype without correcting the growth defect, but overproducing topoisomerase IV, the major cellular decatenase, failed to correct the defects. Our results suggest that type IA topoisomerases play a role in chromosome segregation when gyrase is inefficient. To investigate the role of type IA DNA topoisomerases in genome maintenance, in the third part of the project, we employed genetic approaches combined with suppressor screens, spot assays and microscopy. We found that cells lacking topoisomerase I suffered from supercoiling-dependent growth defects and chromosome segregation defects that could be corrected by deleting recQ, recA or overproducing topoisomerase III and by an oriC15::aph suppressor mutation isolated in the first part of the project. Cells lacking both type 1A topoisomerases formed very long filaments packed with diffuse and unsegregated DNA. Such phenotypes could be partially corrected by overproducing RNase HI or deleting recA, or by suppressor mutations isolated in the first part of the project, that affected cSDR (dnaT18::aph and rne59::aph). Thus, in E. coli, type IA DNA topoisomerases play a role in genome maintenance by inhibiting inappropriate replication from oriC and R-loops and by preventing RecA-dependent chromosome segregation defect through their action with RecQ. The work reported here reveals that inappropriate and unregulated replication is a major source of genome instability. Preventing such replication will ensures proper chromosome segregation leading to a stable genome. RNase HI and type IA DNA topoisomerases play a leading role in preventing unregulated replication. RNase HI achieves this role by modulating ATP dependent gyrase activity and by preventing replication from R-loops (cSDR). Type IA DNA topoisomerases ensure the maintenance of a stable genome by preventing inappropriate replication from oriC and R-loops and by acting with RecQ to prevent RecA dependent-chromosome segregation defects.

The stringent response is a highly conserved physiological response mounted by bacteria under stress (Ojha and Chatterji, 2001; Magnusson et al., 2005; Srivatsan and Wang, 2007; Potrykus and Cashel, 2008). Until recently, the only known players in this pathway were the (p)ppGpp synthesizing and hydrolyzing long RSH enzymes (Mittenhuber, 2001; Atkinson et al., 2011) - RelA and SpoT in Gram negative bacteria and the bifunctional Rel in Gram positive bacteria including mycobacteria. The existence of Short Alarmone Synthetases (SAS) (Lemos et al., 2007, Nanamiya et al., 2008; Das et al., 2009; Atkinson et al., 2011) and Short Alarmone Hydrolases (SAH) (Sun et al., 2010, Atkinson et al., 2011), small proteins possessing a single functional (p)ppGpp synthetase or hydrolase domain respectively, is a recent discovery that has modified this paradigm. Around the same time that the presence of the SAS proteins was reported, we chanced upon such small (p)ppGpp synthetases in the genus Mycobacterium. The stringent response in the soil saprophyte Mycobacterium smegmatis was first reported by Ojha and co-workers (Ojha et al., 2000), and the bifunctional RSH, RelMsm, responsible for mounting the stringent response in this bacterium, has been characterized in detail (Jain et al., 2006 and 2007). RelMsm was the only known RSH enzyme present in M. smegmatis, and consequently, a strain of M. smegmatis deleted for the relMsm gene (ΔrelMsm) (Mathew et al., 2004), was expected to show a null phenotype for (p)ppGpp production. In this body of work, we report the surprising observation that the M. smegmatis ΔrelMsm strain is capable of synthesizing (p)ppGpp in vivo. This unexpected turn of events led us to the discovery of a second (p)ppGpp synthetase in this bacterium. The novel protein was found to possess two functional domains – an RNase HII domain at the amino-terminus, and a (p)ppGpp synthetase or RSD domain at the carboxy-terminus. We have therefore named this protein ‘MS_RHII-RSD’, indicating the two activities present and identifying the organism from which it is isolated. Orthologs of this novel SAS protein occur in other species of mycobacteria, both pathogenic and non-pathogenic. In this study, we report the cloning, purification and in-depth functional characterization of MS_RHII-RSD, and speculate on its in vivo role in M. smegmatis. Chapter 1 reviews the available literature in the field of stringent response research and lays the background to this study. A historical perspective is provided, starting with the discovery of the stringent response in bacteria in the early 1960s, highlighting the development in this area till date. The roles played by the long and short RSH enzymes, ‘Magic Spot’ (p)ppGpp, the RNA polymerase enzyme complex, and a few other RNA and proteins are described, briefly outlining the inferences drawn from recent global gene expression and proteomics studies. The chapter concludes with a description of the motivation behind, and the scope of the present study. Chapter 2 discusses the in vivo and in silico identification of MS_RHII-RSD in M. smegmatis. Experiments performed for the genotypic and phenotypic revalidation of M. smegmatis ΔrelMsm strain are described. Detailed bioinformatics analyses are provided for the in silico characterization of MS_RHII-RSD in terms of its domain architecture, in vivo localization, and protein structure prediction. A comprehensive list of the mycobacterial orthologs of MS_RHII-RSD from a few representative species of infectious and non-infectious mycobacteria is included. Chapter 3 summarizes the materials and methods used in the cloning, purification, and the biophysical and biochemical characterization of full length MS_RHII-RSD and its two domain variants – RHII and RSD, respectively. A detailed description of the purification protocols highlighting the specific modifications and changes made is given. Peptide mass fingerprinting to confirm protein identity, as well as preliminary mass spectrometric, chromatographic, and circular dichroism-based characterization of the proteins under study is also provided. Chapter 4 deals in detail with the in vivo and in vitro functional characterization of the RNase HII and (p)ppGpp synthesis activities of full length MS_RHII-RSD and its two domain variants - RHII and RSD, respectively. The RNase HII activity is characterized in vivo on the basis of a complementation assay in an E. coli strain deleted for the RNase H genes; while in vitro characterization is done by performing a FRET-based assay to monitor the degradation of a RNA•DNA hybrid substrate in vitro. The (p)ppGpp synthesis activity is characterized in terms of the substrate specificity, magnesium ion utilization, and a detailed analysis of the kinetic parameters involved. A comparison of the (p)ppGpp synthesis activity of MS_RHII-RSD vis-à-vis that of the classical RSH protein, RelMsm, is also provided. Inferences drawn from (p)ppGpp hydrolysis assays and the in vivo expression profile of MS_RHII-RSD in M. smegmatis wild type and ΔrelMsm strains are discussed. Based on the results of these functional assays, a model is proposed suggesting the probable in vivo role played by MS_RHII-RSD in M. smegmatis. Chapter 5 describes the attempts at generating MS_RHII-RSD overexpression and knockout strains in M. smegmatis, using pJAM2-based mycobacterial expression system, and mycobacteriophage-based specialized transduction strategy, respectively. The detailed methodology and the principle behind the techniques used are explained. The results obtained so far, and the future work and strain characterization to be carried out in this respect are discussed. Chapter 6 takes a slightly different route and summarizes the work carried out in characterizing the glycopeptidolipids (GPLs) from M. smegmatis biofilm cultures. A general introduction about the mycobacterial cell wall components, with special emphasis on GPLs, is provided. The detailed protocols for chemical composition and chromatographic analyses are mentioned, and the future scope of this work is discussed. Appendix-1 briefly revisits the preliminary studies performed to determine the pppGpp binding site on M. smegmatis RNA polymerase using a mass spectrometry-based approach. Appendices-2, 3, 4 and 5 give a comprehensive list of the bacterial strains; PCR primers; antibiotics, buffers and media used; and the plasmid and phasmid maps, respectively.

Objectives: To assess the expression of the symptoms of jaw muscle pain and its association with alterations in biochemistry, other symptoms and the carriage of staphylococci. Methods: Three different study populations were assessed. The first was selected and examined by the author and consisted of 43 pain and 41 age and sex matched controls. The second was a study of CFS patients who were blinded to the author and the author subsequently examined the associations between jaw muscle symptom reporting and the standardised biochemistry measures. The third study was also blinded to the author but included an investigation of staphylococci and certain cytokine and biochemistry measures. Results: The three studies clearly establish an association between the carriage of toxicogenic coagulase negative staphylococci and the expression of jaw muscle pain in both males and females. These associations were homogeneous and were found whether the patients were selected on the basis of having jaw muscle pain or selected from within a population of patients selected on the basis of having Chronic Fatigue Syndrome. The studies associated the changes with variations in biochemistry and these were in turn associated with symptom expression within the jaw muscle pain patients. These biochemical alterations included the dysregulation of immune cell counts, cytokines, electrolyte and protein metabolism. These symptoms and biochemical changes were associated with pain severity and illness duration and staphylococcal toxin production. From the data a model was developed which shows the mechanisms involved in the development of chronic pain in the jaw muscles. Conclusions: The carriage of toxicogenic coagulase-negative staphylococci were found to be associated with the expression of jaw muscle pain and the alterations in biochemistry associated with these symptoms.

Objectives: To assess the expression of the symptoms of jaw muscle pain and its association with alterations in biochemistry, other symptoms and the carriage of staphylococci. Methods: Three different study populations were assessed. The first was selected and examined by the author and consisted of 43 pain and 41 age and sex matched controls. The second was a study of CFS patients who were blinded to the author and the author subsequently examined the associations between jaw muscle symptom reporting and the standardised biochemistry measures. The third study was also blinded to the author but included an investigation of staphylococci and certain cytokine and biochemistry measures. Results: The three studies clearly establish an association between the carriage of toxicogenic coagulase negative staphylococci and the expression of jaw muscle pain in both males and females. These associations were homogeneous and were found whether the patients were selected on the basis of having jaw muscle pain or selected from within a population of patients selected on the basis of having Chronic Fatigue Syndrome. The studies associated the changes with variations in biochemistry and these were in turn associated with symptom expression within the jaw muscle pain patients. These biochemical alterations included the dysregulation of immune cell counts, cytokines, electrolyte and protein metabolism. These symptoms and biochemical changes were associated with pain severity and illness duration and staphylococcal toxin production. From the data a model was developed which shows the mechanisms involved in the development of chronic pain in the jaw muscles. Conclusions: The carriage of toxicogenic coagulase-negative staphylococci were found to be associated with the expression of jaw muscle pain and the alterations in biochemistry associated with these symptoms.

Following the discovery of the one-dimensional sequence of human DNA, much focus has been directed on microscopy and molecular techniques to learn about the spatial organization of chromatin in a 3D cell. The development of these powerful tools has enabled high-resolution, genome-wide analysis of chromosome structure under many different conditions. In this thesis, I focus on how the organization of interphase chromatin is established and maintained following mitosis. Mitotic chromosomes are folded into helical loop arrays creating short and condensed chromosomes, while interphase chromosomes are decondensed and folded into a number of structures at different length scales ranging from loops between CTCF sites, enhancers and promoters to topologically associating domains (TADs), and larger compartments. While the chromatin organization at these two very different states is well defined, the transition from a mitotic to interphase chromatin state is not well understood. The aim of this thesis is to determine how interphase chromatin is organized following mitotic chromosome decondensation and to interrogate factors potentially responsible for driving the transition. First, I determine the temporal order with which CTCF-loops, TADs, and compartments reform as cells exit mitosis, revealing a unique structure at the anaphase-telophase transition never observed before. Second, I test the role of transcription in reformation of 3D chromosome structure and show that active transcription is not required for the formation of most interphase chromatin features; instead, I propose that transcription relies on the proper formation of these structures. Finally, I show that RNA in the interphase nucleus can be degraded with only slight consequences on the overall chromatin organization, suggesting that once interphase chromatin structures are achieved, the structures are stable and RNA is only required to reduce the mixing of active and inactive compartments. Together, these studies further our understanding of how interphase structures form, how these structures relate to functional activities of the interphase cell, and the stability of chromatin structures over time.