Les topoisomérases (topos) de type IA jouent un rôle primordial dans le maintien et l’organisation du génome. Cependant, les mécanismes par lesquels elles contrôlent cette stabilité génomique sont encore à approfondir. Chez E. coli, les deux principales topoisomérases de type IA sont la topo I (codée par le gène topA) et la topo III (codée par le gène topB). Il a déjà été montré que les cellules dépourvues des topos I et III formaient de très longs filaments dans lesquels les chromosomes ne sont pas bien séparés. Comme ces défauts de ségrégation des chromosomes sont corrigés par l’inactivation de la protéine RecA qui est responsable de la recombinaison homologue, il a été émis comme hypothèse que les topoisomérases de type IA avaient un rôle dans la résolution des intermédiaires de recombinaison afin de permettre la séparation des chromosomes. D’autre part, des études réalisées dans notre laboratoire démontrent que le rôle majeur de la topoisomérase I est d’empêcher la formation des R-loops durant la transcription, surtout au niveau des opérons rrn. Ces R-loops on été récemment identifiés comme des obstacles majeurs à l’avancement des fourches de réplication, ce qui peut provoquer une instabilité génomique. Nous avons des évidences génétiques montrant qu’il en serait de même chez nos mutants topA. Tout récemment, des études ont montré le rôle majeur de certaines hélicases dans le soutien aux fourches de réplication bloquées, mais aussi une aide afin de supprimer les R-loops. Chez E. coli, ces hélicases ont été identifiées et sont DinG, Rep et UvrD. Ces hélicases jouent un rôle dans la suppression de certains obstacles à la réplication. Le but de ce projet était de vérifier l’implication de ces hélicases chez le mutant topA en utilisant une approche génétique. Étonnamment, nos résultats montrent que la délétion de certains de ces gènes d’hélicases a pour effet de corriger plutôt que d’exacerber des phénotypes du mutants topA qui sont liés à la croissance et à la morphologie des nucléoides et des cellules. Ces résultats sont interprétés à la lumière de nouvelles fonctions attribuées aux topoisomérases de types IA dans la stabilité du génome. / Type 1A topoisomerases (topos) play a vital role in the maintenance and organization of the genome. However, the mechanisms by which they control genome stability still remain to be explored. In E. coli, the two type IA topoisomerases are topo I (encoded by topA) and topo III (encoded by topB). It has been shown that cells lacking topo I and III form very long filaments in which the chromosomes are not well separated. As the chromosome segregation defects are corrected by inactivation of the RecA protein, that is responsible for homologous recombination, it has been hypothesized that type IA topoisomerases have a role in the resolution of recombination intermediates to allow chromosome segregation. On the other hand, studies in our laboratory have shown that the major role of topoisomerase I is to prevent the formation of R-loops during transcription, especially at the rrn operons. These R-loops have been recently identified as major roadblocks to the progression of replication forks, which can cause genomic instability. We have genetic evidence suggesting similar effects may occur in our topA mutants. More recently, studies have shown the important role of certain helicases in eliminating roadblocks for replication forks that could sometimes be R-loops. In E. coli, these helicases have been identified and they are DinG, Rep and UvrD. The purpose of this project was to study the roles of these helicases in our topA mutant, using a genetic approach. Surprisingly, our results show that deletions of some of these genes have the effect of correcting rather than exacerbating topA mutant phenotypes that are related to the growth and cell and nucleoid morphology. These results are interpreted in the light of new functions assigned to the type IA topoisomerases in genome stability.

Molecular biophysics is a rapidly evolving field aimed at the physics-based investigation of the biomolecular processes that enable life. In this thesis, we explore two such processes: the thermodynamics of DNA bending, and the mechanism of the RecQ DNA helicase. A computational approach using a coarse-grained model of DNA is employed for the former; an experimental approach relying heavily on single-molecule fluorescence for the latter. There is much interest in understanding the physics of DNA bending, due to both its biological role in genome regulation and its relevance to nanotechnology. Small DNA bending fluctuations are well described by existing models; however, there is less consensus on what happens at larger bending fluctuations. A coarse-grained simulation is used to fully characterize the thermodynamics and mechanics of duplex DNA bending. We then use this newfound insight to harmonize experimental results between four distinct experimental systems: a 'molecular vise', DNA cyclization, DNA minicircles and a 'strained duplex'. We find that a specific structural defect present at large bending fluctuations, a 'kink', is responsible for the deviation from existing theory at lengths below about 80 base pairs. The RecQ DNA helicase is also of much biological and clinical interest, owing to its essential role in genome integrity via replication, recombination and repair. In humans, heritable defects in the RecQ helicases manifest clinically as premature aging and a greatly elevated cancer risk, in disorders such as Werner and Bloom syndromes. Unfortunately, the mechanism by which the RecQ helicase processes DNA remains poorly understood. Although several models have been proposed to describe the mechanics of helicases based on biochemical and structural data, ensemble experiments have been unable to address some of the more nuanced questions of helicase function. We prepare novel substrates to probe the mechanism of the RecQ helicase via single-molecule fluorescence, exploring DNA binding, translocation and unwinding. Using this insight, we propose a model for RecQ helicase activity.

Biomolecular interactions, including protein-protein, protein-DNA, and protein-ligand interactions, are of special importance in all biological systems. These interactions may occer during the loading of biomolecules to interfaces, the translocation of biomolecules through transmembrane protein pores, and the movement of biomolecules in a crowded intracellular environment. The molecular interaction of a protein with its binding partners is crucial in fundamental biological processes such as electron transfer, intracellular signal transmission and regulation, neuroprotective mechanisms, and regulation of DNA topology. In this dissertation, a customized surface plasmon resonance (SPR) has been optimized and new theoretical and label free experimental methods with related analytical calculations have been developed for the analysis of biomolecular interactions. Human neuroglobin (hNgb) and cytochrome c from equine heart (Cyt c) proteins have been used to optimize the customized SPR instrument. The obtained Kd value (~13 µM), from SPR results, for Cyt c-hNgb molecular interactions is in general agreement with a previously published result. The SPR results also confirmed no significant impact of the internal disulfide bridge between Cys 46 and Cys 55 on hNgb binding to Cyt c. Using SPR, E. coli topoisomerase I enzyme turnover during plasmid DNA relaxation was found to be enhanced in the presence of Mg2+. In addition, a new theoretical approach of analyzing biphasic SPR data has been introduced based on analytical solutions of the biphasic rate equations. In order to develop a new label free method to quantitatively study protein-protein interactions, quartz nanopipettes were chemically modified. The derived Kd (~20 µM) value for the Cyt c-hNgb complex formations matched very well with SPR measurements (Kd ~16 µM). The finite element numerical simulation results were similar to the nanopipette experimental results. These results demonstrate that nanopipettes can potentially be used as a new class of a label-free analytical method to quantitatively characterize protein-protein interactions in attoliter sensing volumes, based on a charge sensing mechanism. Moreover, the molecule-based selective nature of hydrophobic and nanometer sized carbon nanotube (CNT) pores was observed. This result might be helpful to understand the selective nature of cellular transport through transmembrane protein pores.