DNA repair

A variety of exogenous and endogenous agents constantly damage the genome. Among the most cytotoxic and genotoxic injuries, double-strand breaks (DSBs) have dramatic outcomes for the cell and their progeny. Failure to repair such lesions leads either to cell death or genomic instability and associated disease phenotypes as illustrated by model organisms where mutations in DNA repair genes lead to cancer predisposition.

To cope with DSBs, cells set up several mechanisms that together compose the DNA damage response (DDR). The DDR must detect, signal and repair DSBs. Eukaryotic cells have evolved two main repair pathways to repair DSBs: non-homologous end-joining (NHEJ) and homologous recombination (HR).

Combining genetics, advanced microscopy and image analysis, we study different steps of the DDR in relation to nuclear organization in budding yeast (Batté et al., 2017; Miné-Hattab et al., 2017) and more recently in human cells. Taking advantage of our findings that the silencing factor Sir3 promotes silent chromatin spreading and telomere clustering independently (Ruault et al, 2011; see project 1), we showed that these two functions of Sir3 contribute to avoid loss of genetic information upon subtelomeric double strand breaks by favouring faithful recombination events between subtelomeres. These findings could be specifically relevant in yeast quiescent cells (see project 3).

In response to double strand breaks (DSB), repair proteins accumulate at damaged sites, forming membrane-less sub-compartments or “foci”. Several models have been intensively debated in the literature, including the liquid phase separation nature of these sub-compartments. However, experimental evidences are often at the limit of the optical resolution.

Combining single molecule microscopy and statistical analysis, we compared the dynamics of several repair proteins accumulating at DNA breaks to elucidate their structure, dynamics and physical nature in living cells. We showed that while most Rfa1 molecules are bound to the ssDNA, Rad52 molecules are free to explore the entire focus reflecting the existence of a liquid droplet around damaged  DNA (Miné-Hattab et al., 2021, Heltberg et al, 2021).

We are currently developing new approaches to visualize key HR events in living cells, including the dynamics of the nucleofilament formed by Rad51 on ssDNA that undergoes the homology search.