No description available.

No description available.

An n-string tangle is a three dimensional ball with n-strings properly embedded in it. In late the 80's, C. Ernst and D. Sumners introduced a tangle model of protein-DNA complexes. This model assumes that the protein is a 3-dimensional ball and the protein-bound DNA are strings embedded inside the ball. Originally the tangle model was applied to proteins such as Cre recombinate which binds two DNA segments. The protein breaks and rejoins the DNA segments and then creatss knotted DNA. When this kind of protein complex bounds circular DNA, there will be two DNA loops outside of the DNA-protein complex. Hence we can use a 2-string tangle model for this complex. More recently, Pathania, Jayaram and Harshey predicted that the topological structure within the Mu protein complex consists of three DNA segments containing five crossigs. Since Mu binds DNA sequences at 3 sites, the Mu protein-DNA complex can be modeled by a 3-string tangle. Darcy, Leucke and Vazquez analyzed Pathania et al's experimental results by using 3-tangle analysis. Based on the 3-string tangle analysis of Mu protein-DNA complex, we addressed the possibility that a protein binds DNA sequences at four sites. Such a protein complex bound to a circular DNA molecule is modeled by a 4-string tangle with four loops outside of the tangle. In this thesis, we decided a biologically relevant 4-string tangle model. We also developed mathematics for solving tangle equations to predict the topology of DNA within the protein complex.

This report combines a series of papers to trace progression in the area of type-2 topoisomerase research. First, Deibler et al. show that knotted DNA is harmful to cells. Knots can block both transcription and replication, and can also act as a catalyst for mutation. Despite the fact that type-2 topoisomerases perform the important functions of unknotting and decatenating DNA, the mechanism by which they accomplish this task is still unknown. Buck and Zechiedrich propose a model in which the enzyme uses local geometry to infer global topology, and thus where to perform segment passage in order to obtain the desired results. In two articles, Liu et al. evaluate this theory quantitatively for the decatenation and unknotting problems. In both cases it is shown that the presence of certain juxtapositions is strongly correlated with global topology. This correlation is not enough, however, and Liu et al. go on to show that when segment passage operations designed to mimic type-2 topoisomerase action are performed at hooked juxtapositions, the overwhelming tendency is towards unknotting and decatenation. / text

The traditional skein relation for the Alexander polynomial involves an oriented knot, K+, with a distinguished positive crossing; a knot K−, obtained by changing the distinguished positive crossing of K+ to a negative crossing; and a link K0, the orientation preserving resolution of the distinguished crossing. We refer to (K+,K−,K0) as the oriented skein triple. A tangle is defined as a pair (B, t) of a 3-dimensional ball B and a collection of disjoint, simple, properly embedded arcs, denoted t. DeWitt Sumners and Claus Ernst developed the tangle model which uses the mathematics of tangles to model DNA-protein binding. The protein is seen as the 3-ball and the DNA bound by the protein as properly embedded curves in the 3-ball. Topoisomerases are proteins that break one segment of DNA allowing a DNA segment to pass through before resealing the break. Effectively, the action of these proteins can be modeled as K− ↔ K+. Recombinases are proteins that cut two segments of DNA and recombine them in some manner. While recombinase local action varies, most are mathematically equivalent to a resolution, i.e. K± ↔ K0. The oriented triple is now viewed as K− = circular DNA substrate, K+ = product of topoisomerase action, K0 = product of recombinase action. The theorem stated in this dissertation gives a relationship between two 2-bridge knots, K+ and K−, that differ by a crossing change and a link, K0 created from the oriented resolution of that crossing. We apply this theorem to difference topology experiments using topoisomerase proteins to study SMC proteins. In recent years, link homology theories have become a popular invariant to develop and study. One such invariant knot Floer homology, was constructed by Peter Ozsváth, Zoltán Szabó, and independently Jacob Rasmussen, denoted by HFK. It is also a refinement of a classical invariant, the Alexander polynomial. The study of DNA knots and links are of great interest to molecular biologists as they are present in many cellular process. The variety of experimentally observed DNA knots and links makes separating and categorizing these molecules a critical issue. Thus, knowing the knot Floer homology will provide restrictions on knotted and linked products of protein action. We give a summary of the combinatorial version of knot Floer homology from known work, providing a worked out example. The thesis ends with reviewing knot Floer homology properties of three particular sub-families of biologically relevant links known as (2, p)- torus links, clasp knots and 3-strand pretzel links.

Diamidines are a class of compounds that target the minor groove of DNA and have antiparasitic and antimicrobial properties. Their mechanism of action has not been fully elucidated, but may include changes in DNA topology. In this study we have investigated such changes using methods of gel electrophoresis including ligation ladders and cyclization assays. We found that topology changes were sequence dependent. Compounds typically caused non-anomalously migrating ATATA sequences to migrate as if they were bent, while A5 sequences that normally migrated anomalously became less so in the presence of certain diamidines. Select compounds induced changes in cyclization efficiency that were also sequence dependent; DB75 significantly abolished cyclization in A5 containing sequences but enhanced it in sequences containing ATATA sites.

The clearance of viruses is largely dependent upon the activation of T cells to generate a robust immune response. However, host responses are suppressed during chronic viral infections. In this thesis, we explored the role of Top2α in DNA topology in individuals with chronic HBV, HCV, and HIV infections. We found that Top2α protein expression and activity were low in T cells derived from chronically virus-infected individuals compared to healthy subjects. Using CD4+ T cells treated with Top2α inhibitor or poisoner as a model, we demonstrated that Top2α inhibition disrupts the DNA topology, suppresses DNA repair kinase (ATM), and telomere protein (TRF2) expression, and induces T cell dysfunction. These findings reveal that Top2α inhibition is a mechanism by which viruses evade the host responses and establish persistent infection, and thus, restoring Top2α levels could be a way of boosting immune responses during chronic viral infections.

No description available.

Des liens de caténation sont progressivement crées lors de la réplication de l’ADN et sont responsables de la cohésion des chromatides sœurs. La topoisomérase IV est une topoisomérase de type II impliquée dans la résolution de ces liens de caténation accumulés derrière la fourche de réplication, et lors de la dernière étape de séparation des chromatides sœurs à la fin de la réplication. Nous avons étudié la liaison de la topoIV à l’ADN ainsi que son activité catalytique à l’aide de méthodes de biologie moléculaire et de génomique. Une expérience de ChIPseq a révélé que l’interaction de la topoIV de chez E.coli avec l’ADN est contrôlée par la réplication. Durant la réplication, la topoIV a accès à des centaines de sites sur l’ADN mais ne se lie qu’à quelques sites où elle exerce son activité catalytique. La conformation locale de la chromatine et l’expression des gènes influencent la sélection de certains sites. De plus, une forte liaison et une activité catalytique renforcée a été trouvée au site de résolution des dimers, dif. Le site dif est situé à l’opposé de l’origine de réplication dans le macrodomaine ter. Nous avons montré qu’il existe une interaction physique et fonctionnelle entre la topoIV et la recombinase XerCD, qui agit au site dif. Cette interaction est médiée par MatP, une protéine essentielle dans l’organisation du macrodomaine ter. L’ensemble de ces résultats montre que la topoIV, XerCD/dif et MatP œuvrent ensemble pour permettre l’étape finale de ségrégation des chromosomes lors du cycle cellulaire. / Catenation links between sister chromatids are formed progressively during DNA replication and are involved in the establishment of sister chromatid cohesion. Topo IV is a bacterial type II topoisomerase involved in the removal of catenation links both behind replication forks and after replication during the final separation of sister chromosomes. We have investigated the global DNA-binding and catalytic activity of Topo IV in E. coli using genomic and molecular biology approaches. ChIP-seq revealed that Topo IV interaction with the E. coli chromosome is controlled by DNA replication. During replication, Topo IV has access to most of the genome but only selects a few hundred specific sites for its activity. Local chromatin and gene expression context influence site selection. Moreover strong DNA-binding and catalytic activities are found at the chromosome dimer resolution site, dif, located opposite the origin of replication. We reveal a physical and functional interaction between Topo IV and the XerCD recombinases acting at the dif site. This interaction is modulated by MatP, a protein involved in the organization of the Ter macrodomain. These results show that Topo IV, XerCD/dif and MatP are part of a network dedicated to the final step of chromosome management during the cell cycle.

Packaging of genomic DNA by proteins and super coiling into chromatin and chromatin-like structures (in bacteria) influences nearly all nuclear process such as replication, transcription, repair, and recombination. A ubiquitous class of enzymes termed “DNA topoisomerases” pay key roles during these process. The reactions catalyzed by the members of the DNA topoisomerases family share a common chemistry, which involves phosphodiester bond breakage and re-joining, to bring about a change in the linking number of DNA. Nevertheless, the underlying mechanisms used by these enzymes differ significantly from another. Consequently, DNA topoisomerases are divided into type I and type II enzymes. The mechanism(s) by which DNA topoisomerases perform their functions, and act as targets for anti-bacterial and anti-neoplastic drugs, has attracted considerable interest. Based on these and other finding, I have chosen DNA gyrase from mycobacteria as the subject of my Ph.D. theses investigation. The prokaryotic enzyme, DNA gyrase, is unique amongst all topoisomerases being the only enzyme capable of introducing negative super coils in to duplex DNA. Since no equivalent enzymatic activity has been reported in humans, this essential enzyme has been exploited as a during target against many microbial infections including tuberculosis.DNA gyrase is a tetrameric protein, comprised of two pairs of subunits, encoded by gyrA and gyrB. Inhibitors of DNA gyrase know till date target either of the two subunits and are categorized broadly in to two class, viz. coumarins and quinolones. With the emergence of multiple-drug resistant strains of pathogenic bacteria such as Mycobacterium tuberculosis, which is a leading cause of death world-wide, there is a need to develops new lead molecules with novel mechanisms of inhibition. Towards this end, a new approach to inhibit the mycobacterial DNA gyrase using single-chain antibody has been explore in the present study. In addition to this, the differences in the catalytic properties of the subunits and assembly of the Mycobacterium smegmatis enzyme vis-à-vis Escherichia coli DNA gyrase have been examined. Further, the in vivo relationship of DNA gyrase with the transcription machinery of the cell has also been investigated, with an emphasis on the biology of mycobacteria.