Séminaire Conjoint Départements de Mathématiques Appliquées et de Biologie - Dr Meriem EL KAROUI
le 22 juin 2015 à 11h00
Dr. Meriem EL KAROUI, University of Edinburgh, Institute of Cell Biology and SynthSys
DNA DOUBLE STRAND BREAK REPAIR IN SINGLE E. COLI CELLS
It has been shown in recent years that even genetically identical cells behave differently because many central processes involve molécules present in small numbers. The inherent randomness of chemical réactions then generates spontaneous fluctuations that can enslave all dépendent processes. Double strand breaks are one of the most deleterious types of DNA damage because they lead death if not repaired. In _Escherichia coli_, the main repair pathway involves the multifunctional RecBCD enzyme, which salvages broken chromosomes by catalyzing the first step of homologous recombination. RecBCD is a heterotrimeric complex that is reportedly present in very low numbers in bacterial cells. This should lead to spontaneous fluctuations in RecBCD levels and non-genetic heterogeneity in the population. Qualitative studies based on Population averages show that bacterial cells that do not express RecBCD are Barely viable, while over-expression of the RecBCD protein leads to less efficient DNA repair. This suggests that the level of RecBCD expression needs to be tightly controlled, and raises the question of how bacterial cells cope with potentially large cell-to-cell fluctuations in this complex.
We have quantified the cell-to-cell variability of RecBCD transcription using chromosomal transcriptional fusions and smFISH and observed significant fluctuations that are consistent with very low levels of transcription. We developed a microfluidic device, combined with highly inclined illumination using laser excitation, which allows us to detect single molecules of GFP _in vivo_. Using translational fusions of RecBCD
to GFP we have shown that RecBCD is present in less than 10 molecules per cell. Surprisingly, the level of fluctuations of each protein of the complex is lower than predicted by stochastic modeling. This suggests that a previously unknown regulatory network is controlling RecBCD expression to avoid potentially harmful fluctuations in RecBCD copy number.
Lieu(x) : Ecole Polytechnique, Amphithéâtre Becquerel
Contact : Yves Mechulam (yves.mechulam at polytechnique.edu)