Orateur
Description
Maintaining genomic integrity is of the highest importance to an organism. Poly-ADP-ribose polymerase 1 (PARP1) is a key actor of the DNA damage response of the cell. Recruited very early at DNA lesions, PARP1 catalyzes the grafting of poly-ADP-ribose (PAR) polymers which signal the presence of the lesions for subsequent repair proteins and trigger chromatin remodeling to facilitate access to DNA breaks. If PARP1 recruitment at DNA lesions is important for repair, its timely release is also crucial since trapping PARP1 at sites of damage via PARP inhibitors used as anticancer drugs was shown to prevent efficient DNA repair. In order to assess in living cells the transient interactions of PARP1 with DNA lesions, we combine fluorescence fluctuation spectroscopy with laser micro-irradiation. Using single-point fluorescence correlation spectroscopy (FCS), we are able to recover association and dissociation rates of repair factors displaying a rapid turnover at DNA lesions. However, for proteins with slower binding kinetics such as PARP1, it is difficult to disentangle binding/unbinding events from the motions of the underlying chromatin in single-point FCS data. To better distinguish both components, we have initiated the development of line-scan FCS approaches that allow probing variable nuclear volumes and performing spatial pair-correlation analyses. We will present our initial results obtained using these methodologies.
| Choix de session parallèle | 2.4 Fluctuation et biologie |
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