Maxime Dahan (Institut Curie)

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Séminaire du laboratoire Gulliver
Contact : Mathilde Reyssat
mathilde.reyssat (arobase) espci.fr

25 novembre 2013 11:15 » 12:15 — Bibliothèque PCT - F3.04

Probing the target search of individual DNA-binding proteins in mammalian cells

For many cellular functions, DNA-binding proteins (DBPs) need to find specific target sites in the genome. Facilitated diffusion (FD), namely the combination of one-dimensional motion along non-specific DNA and three-dimensional exploration, is the dominant model for the target search (TS) of DBPs. Yet, this model has hardly been tested in vivo, particularly in the complex environment of a mammalian nucleus, and it is still controversial whether it accelerates association to specific DNA binding sites. To address that question, we have implemented a TS assay using human cells with a unique target locus for an inducible exogenous searcher, the tetracycline repressor (TetR). Using single-molecule tracking and in situ biochemical measurements of association kinetics, we directly characterize the mobility of TetR, its transient interaction with non-cognate DNA and the kinetics of binding to the specific locus. Overall, we find that the searcher follows a FD strategy but it does not accelerate its search compared to a 3D diffusion-limited process, due to low association efficiency at the target site. I will discuss the potential implications of our results in the context of gene regulation.

Related publications :

1. Ibrahim Cisse, Ignacio Izeddin, Sebastien Causse, Lydia Boudarene, Adrien Senecal, Leila Muresan, Claire Dugast-Darzacq, Bassam Hajj, Maxime Dahan, Xavier Darzacq, « Real time dynamics of RNA Polymerase II clustering in live human cells », Science (2013) 341, 664-7.

2. M. El Beheiry and M. Dahan, “ViSP : representing single particle localization in three dimensions”, Nature Methods (2013) 10, 689–690.

3. S. Abrahamsson, J. Chen, B. Hajj, S. Stallinga, A. Katsov, J. Wisniewski, G. Mizuguchi, P. Soulle, F. Mueller, C. Dugast Darzacq, X. Darzacq, C.Wu, C. I. Bargmann, D. A. Agard, M. Dahan and M.G.L. Gustafsson, “Fast multi-color 3D imaging using aberration corrected multi-focus microscopy”, Nature Methods (2013) 10(1):60-3.

4. D. Normanno, M. Dahan, and X. Darzacq, “Intra-nuclear mobility and target search mechanisms of transcription factors : a single-molecule perspective on gene expression and regulation”, Biophysica and Bichemica Acta (2012), 1819, 482–493





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