Out of Equilibrium analysis of Chk1 dependent DNA replication regulation in Xenopus early embryos

par Diletta Ciardo

Projet de thèse en Sciences de la vie et de la santé

Sous la direction de Kathrin Marheineke et de Arach Goldar.

Thèses en préparation à Paris Saclay , dans le cadre de Structure et Dynamique des Systèmes Vivants , en partenariat avec Institut de Biologie Intégrative de la Cellule (I2BC) (laboratoire) et de Université Paris-Sud (établissement de préparation de la thèse) depuis le 01-10-2016 .


  • Résumé

    Appréhender l'orchestration des facteurs protéiques qui régulent dans le temps et l'espace, la duplication du génome (réplication de l'ADN) dans le noyau d'une cellule eucaryote est un défi majeur de la biologie moléculaire. Nous avons montré que l'augmentation de la kinase Chk1 (surexpression), inhibe la réplication de l'ADN, cependant, les mécanismes impliqués ne sont toujours pas clairs. Notre projet, à l'interface entre la biologie moléculaire et la physique statistique, vise à identifier de nouveaux mécanismes d'action de Chk1 en utilisant la protéomique quantitative et le peignage moléculaire de l'ADN. Cette technique d'étirement des fibres d'ADN permet d'observer une molécule unique. Elle a déjà montré l'existence d'une grande variabilité de program de réplication entre cellules d'une même population, en partie à cause de la nature stochastique de ce mécanisme. La séquence des événements qui activent ou répriment la réplication est difficile à analyser. Nos nouvelles données expérimentales permettront de développer notre modèle quantitatif qui s'enrichira en parallèle des résultats des simulations numériques. Comprendre comment Chk1 régule la réplication permettra d'expliquer pourquoi les niveaux Chk1 sont augmentés dans certains types de cancers. De plus notre nouvel modèle analytique permettra de relier le programme de réplication de l'ADN d'une cellule unique à celui d'une population de cellules constituant les organismes multicellulaires.

  • Titre traduit

    Out of Equilibrium analysis of Chk1 dependent DNA replication regulation in Xenopus early embryos


  • Résumé

    Our thesis project is an interdisciplinary approach between the fields of experimental molecular biology and biochemistry including single molecule analysis on the one hand and the field of statistical physics and simulation methods on the other hand. The lab of K. Marheineke (I2BC) has a long experience in the DNA replication and DNA combing technique in multicellular organisms whereas Arach Goldar in the team from J. Soutourina (I2BC) has expertise on DNA replication in the unicellular yeast and theoretical models and simulations. New experimental advances have enabled the study of dynamics of replication regulation at the single-molecule levels by DNA combing, a DNA fiber stretching technique. They demonstrate the coordinated activation of several replicons, or replicon clusters and domains which is also controlled by the ATR-Chk1 dependent replication checkpoint that is activated in response to stalled replication forks. The host lab has shown by inhibition or depletion of this essential protein kinase Chk1 that it negatively regulates the replication program at the level of replication clusters and not inside active clusters on single replication origins. In addition the lab showed that modest Chk1 overexpression inhibits origin firing illustrating that Chk1 levels are tightly regulated and are therefore a limiting factor during S phase. Here, we will measure in detail the dynamics of DNA replication as initiation frequencies in an in vitro system of replicating Xenopus nuclei after overexpressing kinase Chk1 by molecular combing of DNA. Next, combining DNA combing, immunoprecipitation and quantitative proteomics the project will identify the interacting partners of Chk1 during DNA replication after Chk1 overexpression. Both, the obtained experimental results of replication dynamics and molecular pathways will be used to build a theoretical model of replication dynamics. The common feature of existing theoretical methods that describe the kinetics of DNA replication is that they do not take into account the details of the dynamics path of this process. By building a macroscopic model of DNA replication without a priori considerations on the processes that induce the duplication of the genome and by using thermodynamics arguments, we will show that the replication process evolves as an out-of-equilibrium phenomenon. We will carry out, the development of a general non-equilibrium framework that will use concepts from equilibrium and non-equilibrium statistical mechanics. In this way we will show that the dynamics of DNA replication could be mapped to a Langevin type equation, whose generic properties will allow us to define the source of entropy production that drives these processes out of equilibrium. The other advantage to map these processes to a dynamics stochastic equation is that we will have the opportunity to make an analogy with the dynamics of an overdumped oscillator. This analogy would help us to define a potential in which the replication process evolve. This potential will allows us to infer from our produced experimental data the local rate of DNA replication, and therefore, to analyze the effect of inhibition/ over expression of Chk1 kinase on the spatio-temporal pattern of DNA replication. We will backup our theoretical studies with numerical ones, where by using dynamical MonteCarlo method we will simulate DNA replication. The expected results of our interdisciplinary project will help to understand why Chk1 is frequently overexpressed in several cancers. We will also present a quantitative model for the dynamics of DNA replication in higher eukaryotes linking the replication program of a single cell to a cell population.