Regulation of Sister-Chromatid Cohesion

Orderly execution of two critical events during the cell cycle--DNA replication and chromosome segregation--ensures the stable transmission of genetic materials. The cohesin complex physically connects sister chromatids during DNA replication in a process termed sister-chromatid cohesion. Timely establishment and dissolution of sister-chromatid cohesion is a prerequisite for accurate chromosome segregation, and is tight regulated by the cell cycle machinery and cohesin-associated proteins. Errors in this process can lead to aneuploidy and promote tumorigenesis. Research in this dissertation has provided several key insights into the regulation of sister-chromatid cohesion during the mitotic cell cycle. First, we report the crystal structure and functional characterization of human Wapl, a key negative regulator of cohesin that promotes cohesin release from chromatin. Our results indicate that Wapl-mediated cohesin release from chromatin requires extensive physical contacts between Wapl and multiple cohesin subunits. Second, we have determined the crystal structure of human SA2-Scc1 cohesin subcomplex, which is the interaction hub for cohesin regulators. Further biochemical and functional analyses reveal the direct competition between Wapl and the cohesion protector Sgo1 for binding to a conserved site on SA2-Scc1. Our results implicate a role for this direct antagonism in centromeric cohesion protection. Third, we report the crystal structure of human Pds5B bound to a conserved peptide motif found in both Wapl and Sororin. Further biochemical and functional studies suggest that Pds5 has both positive and negative roles in cohesion regulation and establish the molecular basis for how Wapl and the cohesin-stabilizing factor Sororin antagonistically influence cohesin dynamics on chromosomes. The structure reveals inositol hexakisphosphate (IP6) as an unexpected cofactor of Pds5. The IP6-binding segment of Pds5B engages the N-terminal region of Scc1 and inhibits the binding of Scc1 to Smc3. Our results suggest a direct role of Pds5 in cohesin release from chromosomes by stabilizing a transient, open state of cohesin during its ATPase cycle. Finally, we show that cohesin loading onto chromosomes requires the phosphorylation of MCM2-7 by Cdc7-Dbf4 kinase (DDK) during early S phase, when a mega-complex composed of MCM2-7, Scc2/4 and cohesin is formed. At active replication forks, inactivation of multiple replisome components impairs cohesin loading, weakens MCM-Scc2/4-cohesin interaction and leads to cohesion defects. By contrast, interfering Okazaki fragment processing and nucleosome assembly during DNA replication do not impact interphase cohesion, suggesting that cohesion establishment occurs before Okazaki fragment maturation and histone deposition. Our results demonstrate that DNA replication-coupled cohesin loading is required for the establishment of sister-chromatid cohesion. In conclusion, combining structural, biochemical and cellular approaches, our studies advance the molecular understanding of spatial and temporal regulation of the establishment and dissolution of sister-chromatid cohesion.