Long Term Friction: from Stick-Slip to Stable Sliding

We have devised an original laboratory experiment where we investigate the frictional behaviour of a single crystal salt slider over a large number of deformation cycles. Because of its physical properties, salt, a surrogate for natural faults, allows for friction and plastic deformation and pressure solution creep to be efficient on the same timescale. During the same experiment, we observe a continuous change of the frictional behaviour of the slider under constant conditions of stiffness, temperature and loading velocity. The stick-slip regime is progressively vanishing, eventually reaching the stable sliding regime. Concomitantly, the contact interface, observed under the microscope, develops a striated morphology with contact asperities increase in length and width, arguing for an increase in the critical slip distance dc. Complementary experiments including velocity jumps show that the frictional parameters of the rate and state friction law, a and b, progressively vanish with the cumulative slip. In our experimental conditions, the ultimate stage of friction is therefore rate and state independent.Comment: 10 pages; 4 figures; 1 Tabl

DNA mechanics as a tool to probe helicase and translocase activity

Helicases and translocases are proteins that use the energy derived from ATP hydrolysis to move along or pump nucleic acid substrates. Single molecule manipulation has proved to be a powerful tool to investigate the mechanochemistry of these motors. Here we first describe the basic mechanical properties of DNA unraveled by single molecule manipulation techniques. Then we demonstrate how the knowledge of these properties has been used to design single molecule assays to address the enzymatic mechanisms of different translocases. We report on four single molecule manipulation systems addressing the mechanism of different helicases using specifically designed DNA substrates: UvrD enzyme activity detection on a stretched nicked DNA molecule, HCV NS3 helicase unwinding of a RNA hairpin under tension, the observation of RecBCD helicase/nuclease forward and backward motion, and T7 gp4 helicase mediated opening of a synthetic DNA replication fork. We then discuss experiments on two dsDNA translocases: the RuvAB motor studied on its natural substrate, the Holliday junction, and the chromosome-segregation motor FtsK, showing its unusual coupling to DNA supercoiling

Etude sur pince magnétique de la jonction de Holliday (les interactions de la jonction avec la protéine MutS ou la protéine RecG)

Ce travail présente l'étude à l'echelle de la molécule unique la jonction de Holliday, et de ses interactions avec les protéines MutS et ReG. Nous commençons par le comportement de la jonction de Holliday. La présence au niveau du centre de la jonction d'une hétérologie entre les séquences suffit à bloquer la migration sous certaines conditions expérimentales. Ce phénomène nous permet de détecter les hétérologies de séquences entre deux molécules. Nous avons étudié l'interaction entre une molécule d'ADN pre sentant une jonction de Holliday, et la protéine MutS. La présence de MutS bloque la migration lorsqu un mésappariement arrive au niveau du centre de la jonction. La protéine RecG est une hélicase qui intervient dans le sauvetage des fourches de réplication. Des études suggèrent sa capacité à faire rebrousser les fourches de réplication arrêtées. Nous avons étudié ces réactions à l'échelle de la molécule unique. Nous avons observé la migration de la jonction. Cela nous a permis d'effectuer une mesure directe de la vitesse de l'enzyme, sa processivité et le couple maximal fourni par l'enzyme. Nous avons étudié la dépendance en concentration d'ATP et d'enzymePARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Transcription par une ARN polymérase (mesures de forces à l'échelle de la molécule unique)

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Mapping of Single-Base Differences between Two DNA Strands in a Single Molecule Using Holliday Junction Nanomechanics

International audienceObjective The aim of this work is to demonstrate a novel single-molecule DNA sequence comparison assay that is purely based on DNA mechanics. Methods A molecular construct that contained the two homologous but non-identical DNA sequences that were to be compared was prepared such that a four-way (Holliday) junction could be formed by the formation of heteroduplexes through the inter-recombination of the strands. Magnetic tweezers were used to manipulate the force and the winding applied to this construct for inducing both the formation and the migration of a Holliday junction. The end-to-end distance of the construct was measured as a function of the winding and was used to monitor the behavior of the Holliday junction in different regions of the intra-molecular recombination. Main Results In the appropriate buffer, the magnet rotation induces the migration of the Holliday junction in the regions where there is no sequence difference between the recombining sequences. In contrast, even a single-base difference between the recombining sequences leads to a long-lasting blockage of the migration in the same buffer; this effect was obtained when the junction was positioned near this locus (the site of the single-base difference) and forced toward the formation of heteroduplexes that comprise the locus. The migration blockages were detected through the identification of the formation of plectonemes. The detection of the presence of sequence differences and their respective mappings were obtained from the series of blockages that were detected. Significance This work presents a novel single-molecule sequence comparison assay that is based on the use of a Holliday junction as an ultra-sensitive nanomechanism; the mismatches act as blocking grains of sand in the Holliday "DNA gearbox". This approach will potentially have future applications in biotechnolog

Etude de MutS à l'échelle de la molécule unique

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Recombinaison génétique à l'échelle de la molécule unique (micromécanique des jonctions de Holliday et activité du complexe RuvAB)

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Evolution expérimentale de bactéries par brassage de génome

Cette thèse porte sur des expériences d'évolution de bactéries, avec le but d explorer expérimentalement comment des transferts horizontaux d ADN au sein d une population de bactéries peuvent ou non modifier l évolution de cette population. La méthode utilisée pour permettre des échanges d ADN à l intérieur de la population repose sur la propriété de transformation naturelle de la bactérie Acinetobacter baylyi. Une expérience pilote sans brassage de génomes a été faite sur 3000 générations et nous avons analysé par séquençage les mutations et la diversité apparues pendant cette expérience. On observe une structure en multiples sous-populations compatible avec un régime d'interférence clonale. Une expérience d évolution avec échange d'ADN ainsi qu'une expérience contrôle avec une bactérie non compétente ont été réalisées dans les mêmes conditions sur 1300 générations. Des séquençages de génomes ont été effectués sur des populations et des clones à différents temps de chaque expérience d'évolution afin de mettre en évidence les mutations apparues ainsi que leur fréquence dans la population. La diversité dans l'expérience d'évolution avec brassage est nettement plus élevée jusqu à 970 générations et est due à différents types de mutations alors que dans l'expérience sans brassage, la diversité est essentiellement due à des SNP. Cependant dans l'expérience avec brassage, on observe à 1300 générations une baisse de la diversité qui est liée à la fixation d'une mutation dans un gène essentiel pour la transformation. Ces résultats préliminaires laissent penser que le brassage a un avantage à court terme mais qu'à long terme l'avantage lié à l'échange s'amenuiseThis thesis focuses on experiments in bacterial evolution, with the aim to explore experimentally how horizontal transfer of DNA within a bacterial population, may or may not modify the evolution of this population. The method used to allow exchanges of DNA within the population, is based on the property of natural transformation of the bacterium Acinetobacter baylyi. A pilot experiment without genome shuffling was made over 3000 generations, and we analyzed by sequencing the mutations and the diversity that emerged during this experiment.There is a structure in multiple actively competing subpopulations that appears consistent with a system of clonal interference. An experimental evolution with exchange of DNA and a control experiment with a non- competent bacterium were performed under the same conditions over 1300 generations. The sequencing of genomes have been conducted on populations and clones at different times of each experiment to detect the mutations that occurred and their frequency in the population. Diversity in the evolution experiment with genome shuffling is significantly higher up to 970 generations and is due to different types of mutations, whereas in the control experiment diversity is mainly due to SNP. However in the experiment with shuffling, we observe a decrease in the diversity at 1300 generations which is due to the fixation of a mutation in a gene essential for transformation. These preliminary results suggest that the genome shuffling has a short term benefit while on the long term the benefit from the exchange is dwindlingPARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF