UC Berkeley

A central problem in biology is how genetic material is organized and inherited during cell divisions without loss of information. The conserved protein complex cohesin functions to ensure chromosomes are partitioned equally when cells divide. Cohesin mediates sister chromatid cohesion, chromosome condensation, efficient repair of DNA damage and regulation of transcription. The activity by which cohesin performs these functions is through DNA tethering. Tethering can occur between two sister chromatids or within a single chromatid to bring together two separate positions along its length. Within this dissertation, I will describe novel insights into the mechanism by which cohesin performs these critical activities on chromosomes. I chose to study this problem in the budding yeast Saccharomyces cerevisiae because of the unparalleled genetic tools available in this model eukaryote.

Cohesin consists of four core subunits referred to in yeast as Mcd1p, Scc3p, Smc1p, and Smc3p, whereas spatiotemporal control of cohesin activity depends on auxiliary factors. To better understand cohesin architecture and regulation, I performed a genetic screen to identify novel mutants of the Smc3p subunit. Characterization of these mutants has revealed functions for previously undescribed regions of Smc3p and new activities for previously described domains that further our understanding of this complex molecular machine. First, I describe a series of mutations at the interface between Smc3p and Mcd1p termed the DNA “exit gate”. This investigation revealed an unexpected role for this interface in loading cohesin on chromosomes. Second, I reveal a novel role for the Smc3p hinge domain in cohesion maintenance that is independent of its ability to stably bind chromosomes. This discovery lends additional weight against a simple “embrace” model for DNA tethering by cohesin still supported by many in this field. Moreover, this observation suggests that communication between the Smc3p hinge, Mcd1p, and regulator Pds5p is required for cohesion maintenance. Finally, I show that a specific region in the middle of the cohesin ring is critical for a step in the process of cohesin loading onto chromosomes. This result is surprising since it suggests that a contortion of the cohesin ring is critical for it to productively bind chromosomes. In sum, this dissertation advances our understanding of the mechanism by which cohesin functions and supports a variation of the “handcuff” model in which an activity after DNA binding is required for cohesin to achieve tethering.