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JF Allemand - associated team

Multiscale physics of living systems

Page of the team on the LPENS web site

Classical biochemical methods have greatly advanced the understanding of fundamental process such as DNA replication, DNA repair, transcription and chromatin remodelling. However these approaches have reached their limits since bulk assays yield the average activity of a population of molecules, ignoring fine details such as molecular variability, the diffusion time needed to reach target etc. These limitations have been overcome with the development of single molecule techniques allowing for example to manipulate and visualize single DNA molecules and their interactions with proteins. In these approaches developed in the last fifteen years, the activity of a single enzyme is recorded in real time. The team lead by David Bensimon and Vincent Croquette has applied these techniques to study the mechanical properties of DNA and investigate its interactions with a variety of proteins, such as the molecular motors involved in DNA replication (e.g. DNA polymerases) and repair (e.g. the UvrD and RecQ helicases).

Significant results.

In magnetic tweezers, a single DNA molecule tethers a small magnetic bead to a glass surface. Magnets placed above the bead pull on the DNA with a constant force while the torsional state of DNA can be easily control through the angular position of the magnets. With this tool, the team has studied the T4 replisome where a helicase separates the two DNA parental strands which are copied by two polymerases. On the leading strand the polymerase copies continuously the DNA in the wake of the helicase while this process is done discontinuously on the lagging strand by a series of Okazaki segments. Each Okazaki segment is initiated by a primase which works in coordination with the helicase. Using magnetic tweezers, we have observed that when the primase initiates a new primer, the helicase continues to unwind DNA and doesn’t pause for the primase to terminate its activity. Since the primase is a rather slow enzyme, we have demonstrated that two situations can occur : in some cases the primase detaches from the helicase and stays bound to its primer while in other cases the two enzymes remain bound and a ssDNA loop is extruded between them by the unwinding action of the helicase. We have also shown that the replicative (gp41) and repair helicases (RecQ) have different unwinding mechanisms : replicative helicases use a passive strategy waiting for DNA spontaneous opening to move in the fork while repair enzymes display an active mechanism destabilizing the duplex. Another activity of the team concerns the active control of proteins at the single cell level. The rational here is to study cell lineage in the course of embryo development, the evolution of a tumor or the plasticity of neurons in the learning phase. To address these questions, we use photo-activable molecules which can be triggered in space and time. The principle is to use caged signalling molecules that upon UV illumination release an active ligand. This ligand then binds to its receptor releasing a fused protein from its complex with cytoplasmic chaperones. These studies are done using zebrafish which as a model vertebrate is transparent, develops rather rapidly and can be easily genetically modified.

Manosas M., Spiering M.M., Zhuang Z., Benkovic S., Croquette V., Coupling DNA unwinding activity with primer synthesis in the bacteriophage T4 primosome. Nature Chemical Biology (2009), 5 — 904 - 912.

Manosas M., Guang Xi X., Bensimon D., Croquette V., Active and passive mechanisms of helicases. NAR (2010), sous presse.

Sinha D.K., Neveu P., Gagey N., Aujard I., Benbrahim-Bouzidi C., Le Saux T., Rampon C., Gauron C., Goetz B., Dubruille S., Baaden M., Volovitch M., Bensimon D., Vriz S., Jullien L., Photocontrol of Protein Activity in Cultured Cells and Zebrafish with One- and Two-Photon Illumination. Chembiochem. (2010), 11(5) — 653-663.

Neveu P., Aujard I., Benbrahim C., Le Saux T., Allemand J.F., Vriz S., Bensimon D., Jullien L., A caged retinoic acid for one- and twophoton excitation in zebrafish embryos. Angew Chem Int Ed Engl (2008), 47(20) — 3744-6.