Date : 01/08/2011

Internship proposal for : Master 1 or Master 2

Laboratory
Centre de Génétique Moléculaire
UPR 3404 CNRS
bat26
1 avenue de la terrasse
91198 Gif sur Yvette
Website : http://www.cgm.cnrs-gif.fr/espeli/index.html
Main discipline : Molecular Biology
Lab director Olivier Espéli

Mentor
Olivier Espeli
email : This e-mail address is being protected from spam bots, you need JavaScript enabled to view it
phone : 33169823214

Subjects
1.: Chromosome organization
2.: sister chromatid cohesion
3.: DNA topology

Tools and methodologies
1.: Whole genome approaches, ChIPseq, Hi-C
2.: molecular biology
3.: superesolution fluorescence microscopy

Summary of lab's interests

In E. coli, following their replication sister chromatids remain colocalized for 20 minutes before their segregation to each daugther cells, this step is called sister chromatid cohesion. In bacteria, little is known about sister chromosome cohesion and its possible functional role. The main goal of our project is to reveal the structuring mechanisms involved in sister-chromatid cohesion in bacteria. Recently, we have demonstrated that topological links (precatenanes) between newly replicated chromatids could promote cohesion of the sister chromatids in bacteria and for some regions of the eukaryotic chromosomes. It is of a particular interest to establish directly if precatenane links are formed during the replication of the E. coli chromosome, if they are maintained for a sufficient long time to allow cohesion and what are the elements involved in the regulation of their removal. We have developed a recombination test to measure precatenation of the chromatids in vivo. To address the question of the spatial organization of the sister chromatids, we are combining the recombination assay and Chromosome Conformation Capture (Hi-C) techniques that allow its characterization at the whole genome wide scale. It gives us the opportunity to survey molecular cohesion on the entire chromosome. To study sister chromatid dynamics at the single cell level we are developing new tools for cellular biology analysis of chromosome. With these tools, we should be able to transiently differentiate the two sisters chromatids, and to analyze precisely their connections with high resolution using FRET assay and super-resolution microscopy.

Summary of project

In any living organism, the control of DNA topology is achieved by the fine tuning of the Topoisomerases activity with the generation of DNA twist introduced by DNA translocating enzymes (RNA and DNA polymerases, helicases...). The mechanisms used by the cell to control of topoisomerase activities are not yet fully understood. We have identified that topological links, formed between sister chromatids behind replication forks, are involved in the process maintaining sister chromatids into close proximity. This observation is puzzling, because it involves an unexpected inhibition of the activity of the Topoisomerase IV on the newly replicated DNA behind the replication fork. We propose, for the internship, a project dealing with the characterization of this control mechanism. According to the main interest of the intern, the project will involve ChIPseq experiments and analysis, fluorescence microscopy with high end techniques such as palm superresolution or classic genetic and molecular biology assays.