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Sujet de stage / Master 2 Internship

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X-ray scattering dynamic study of chromatin conformation during DNA replication

Contact: GOBEAUX Frederic, , frederic.gobeaux@cea.fr, +33 1 69 08 24 74
Summary:
We propose to study the structural modifications of the tridimensional organization of chromatin during cell cycle. For this purpose we will combine small angle x-ray scattering on synchronized yeast cells populations and flow cytometry.
Possibility of continuation in PhD: Oui
Deadline for application:17/03/2019

Full description:
The tridimensional organization of the genome and its dynamics in live cells are decisive to perform its functions. It is crucial to understand them and to identify the parameters controlling them. Current state of the art allows describing the short range (<10 nm) and long range (>250 nm) organization of chromatin conformation in the nucleus. However, there is an intermediate range (10-250 nm) where chromatin organization is difficult to apprehend. This range corresponds to the size of proteic complexes that modify chromatin and harness genome replication.

With this internship, we thus propose to study the relationship between chromatin structure between 1 and 100 nm and the DNA replication dynamics. For this purpose, small angle x-ray scattering (SAXS) will be used to monitor chromatin conformation changes correlated with flux cytometry to follow genome replication. Scattering will be directly obtained from populations of synchronized cells. Concepts from polymer physics will be used to quantify the links between replication dynamics and chromatin structure.

Our model system will be yeast cells, Saccharomyces cerevisiae. Like for any eukaryotic cells, yeast cellular cycle exhibit three phases before division: 1) DNA duplication preparation (G1 phase) 2) DNA duplication (S phase) and 3) Duplicated DNA control and preparation to division (G2 phase). Type A S. cerevisiae cells will be synchronized at the end of their G1 phase using a sexual pheromone called alpha factor. By removing alpha factor through successive washings, the population of cells synchronized at the end of the G1 phase will be released in the S phase. At the same time, SAXS patterns will be collected while samples will be garnered to perform fluorescence activated cell sorting (FACS).

The intership will take place between the Laboratoire Interdisciplinaire sur l'Organisation Nanométrique et Supramoléculaire (Frédéric Gobeaux, Patrick Guenoun) and the Laboratoire Transcription et Génomique (Arach Goldar).
Technics/methods used during the internship:
Cell culture Small angle x-ray scattering Flow cytometry Signal analysis Statistical physics Polymer physics

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