| The evolutionarily-conserved cohesin complex promotes accurate chromosome segregation, the repair of DNA double strand breaks (DSBs), chromosome condensation, and transcriptional regulation. To facilitate these diverse biological functions, cohesin binding to chromatin is regulated both temporally and spatially. Additionally, the chromatin-bound cohesin can be induced to physically tether sister chromatids together. Binding of cohesin to chromatin is not sufficient to tether sister chromatids however. Instead, an additional post-chromatin binding step promoted by the Eco1p acetyl-transferase is required to tether the sister chromatids, a process coined 'cohesion generation'. In this thesis, I define the essential target of the Eco1p acetyl-transferase for DSB-induced cohesion generation, and discover a regulatory cascade that coordinates Eco1p activity with the DNA damage checkpoint in budding yeast. I exploit differences in the composition of the cohesin complex in mitosis and in meiosis to identify the Mcd1p cohesin subunit as the critical target of Eco1p. Furthermore, I provide evidence that Eco1p's ability to acetylate Mcd1p is enhanced by Mcd1p phosphorylation by the DNA damage checkpoint kinase Chk1p. Mcd1p acetylation then acts antagonistically to Wpl1p, a known inhibitor of cohesin generation during S phase, resulting in the generation of DSB-induced cohesion generation. Intriguingly, the Mcdlp modification sites are located in the proximity of the Smc3p ATPase, whose acetylation by Eco1p is required for cohesion generation during S phase. I show that these two targets are similar in their ability to antagonize Wpl1p, but cannot compensate for each other to promote cohesion generation. Furthermore, I show that Eco1p and Wpl1p modulate the Smc3p ATPase by preventing and promoting the binding of ATP, respectively. Finally, I establish a system to understand the hierarchy of cohesin functions. My observations support a model where the majority of cohesin in the cell is dispensable for chromosome segregation but essential for promoting genomic integrity by ensuring accurate DNA damage repair and the stability of repetitive DNA. Altogether, my thesis work has significantly expanded our understanding of how cohesion generation is modulated throughout the cell cycle to facilitate cohesin's segregation and non-segregation functions and provides a framework for future investigations into the molecular basis of sister chromatid cohesion. |
