Abstract
DNA double stranded breaks (DSBs) occur constantly, and are highly deleterious, which is why eukaryotic cells have evolved mechanisms of repair to ensure genomic stability. A crucial aspect of the DNA damage response is recognition of the lesion and activation of the DNA damage checkpoint, which prevents cells from dividing prior to repairing the lesion. Following a DSB in yeast, the two PI3-like protein kinases Mec1 and Tel1 phosphorylate a cascade of downstream effectors that lead to cell cycle arrest. A key target for Mec1 regulated phosphorylation is Rad53, which, following Mec1 mediated phosphorylation undergoes autophosphorylation. Following repair, cells must properly deactivate the checkpoint in order to continue the cell cycle, a process termed recovery. Recovery defective cells fail to deactivate the checkpoint and therefore cannot resume cell cycle progression, despite being able to repair. This study focuses on the role of histone chaperones and histone modifying proteins in the deactivation of the checkpoint, specifically Asf1, CAF-1, Rtt109, and Rtt101. Asf1 and CAF-1 are histone H3-H4 chaperones. Interestingly, Asf1 binds Rad53 prior to suffering damage. Once Rad53 is phosphorylated during the damage response, its affinity to Asf1 is greatly reduced. Rtt109 is a histone acetyl transferase and acetylates H3K56 after Asf1 binds H3, which then weakens Asf1-H3 interactions. Rtt101, a ubiquitin ligase, ubiquitylates H3K56ac to aide in the dissociation of Asf1 from H3. Therefore, the hypothesis proposed in this thesis is that dissociating H3-H4 from Asf1 is necessary to allow Asf1 to interact and sequester Rad53 following repair, thus deactivating the checkpoint and facilitating recovery.