Le rôle de la structure de la chromatine naissante dans la réponse au stress réplicatif

The information necessary for every cellular process is encoded in DNA, and the survival of any species requires that it faithfully and successfully replicates this molecule. Paradoxically, it is when cells undergo DNA replication that the integrity of their genome is the most vulnerable to agents that cause DNA damage. As such, cells possess a wide variety of mechanisms that protect genomic integrity, which includes regulation of chromatin structure. In all eukaryotic species studied so far, newly synthesized nucleosomes in S-phase harbor transient modifications that influence, amongst other things, the response to replicative stress. Particularly, in Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56ac) is added by Rtt109 during DNA replication, and is removed after completion of genome duplication by members of the sirtuin family of histone deacetylases, Hst3 and Hst4. Interestingly, any perturbation of acetylation/deacetylation cycle causes severe growth defects accompanied by an extreme sensitivity to replicative stress-inducing genotoxins. However, the H3K56ac functions underlying these phenotypes are poorly understood.  To further understand the role of H3K56ac in maintaining genomic integrity, we examined the phenotypes caused by constitutive hyperacetylation of H3K56, such as in the hst3Δ hst4Δ mutant. We first characterized the effects of H3K56 hyperacetylation on the DNA damage response (DDR), and found that it induces spontaneous and aberrant appearance of DNA repair protein foci, as well as hyperactive DDR signaling. We next performed a screen to identity histone mutations that can suppress the growth defects of hst3Δ hst4Δ mutants, and found that H3K79 methylation and H4K16 acetylation are toxic for cells harboring hyperacetylated H3K56. Our results suggest that preventing the addition of these two modifications improves the growth of hst3Δ hst4Δ mutants by reducing the activity of Rad53, a key kinase for DDR signaling. Hst3 and Hst4 can be inhibited by nicotinamide (NAM), a pan-sirtuin inhibitor, which induces H3K56 hyperacetylation. We performed a chemogenetic screen to identify genes whose mutation confers fitness defects in the presence of nicotinamide (NAM)-induced H3K56 hyperacetylation. Interestingly, amongst the tops hits were mutants for SLX4 and PPH3, two genes encoding proteins involved in the dampening of DDR signaling. By using slx4Δ and pph3Δ cells to probe for the root causes of sensitivity to DDR signaling in cells with constitutive H3K56ac, we found that the defects of hst3Δ hst4Δ mutants are instigated by Rad53 hyperactivity-mediated inhibition of late replication origin firing and crippling of the response to oxidative stress. Intriguingly, this last screen led to the surprising finding that telomere length influences replication origin firing genome-wide. Moreover, our results suggest that cells with short telomeres are sensitive to NAM and replicative stress because of sequestration of origin firing factors to telomeres.  In summary, results presented herein indicate that hyperactive DDR signaling is a major component of the extreme phenotypes observed in cells harboring constitutive H3K56ac. Collectively, our work unveils novel mechanisms by which chromatin structure influences cells’ ability to cope with replicative stress.

Keyphrases: H3K56ac, DNA damage response, Replicative stress, DNA replication, Chromatin structure, Telomeres, Chromatin assembly