UCSF

Progression through the eukaryotic cell cycle occurs through an exact sequence of precisely coordinated events, culminating in the segregation of the duplicated copies of all the cellular components into two daughter cells. Crucial to the faithful execution of all the cellular events that must take place is the temporal separation of mutually exclusive enzyme activities. Controlling all these processes are a network of regulatory proteins, including a number of kinases and ubiquitin ligases, which ensure the proper sequence of events in every cycle. This thesis explores the control of one particular event in the cell cycle, establishment of sister-chromatid cohesion, using Saccharomyces cerevisiae as a model organism. Cohesion between chromatids is necessary to keep track of which pieces of DNA need to be separated to opposite daughter cells, and also has a function in the molecular recognition of readiness for anaphase. Cohesion establishment is restricted to S phase of a normal cycle and can be reactivated in metaphase upon DNA damage, but the basis for this regulation was unknown. I have found that the phosphorylation-mediated degradation of the cohesion-promoting protein Eco1 is responsible for the differential regulation of cohesion generation over time and in response to stress. Eco1 phosphorylation occurs by an interesting cascade of primed kinases: Cdk1 bound to B-type cyclins phosphorylates Eco1 at a site that creates a consensus motif for the kinase Cdc7-Dbf4, whose activity in turn creates a consensus motif for Mck1, a homolog of the tumor-suppressor GSK-3. Only after all three kinases have phosphorylated Eco1 is it targeted for ubiquitination by SCF-Cdc4. This is because the substrate adapter Cdc4 only binds degrons with precisely-spaced diphosphates. The combination of all these factors is necessary to degrade Eco1 and shut off establishment of new cohesion, setting up a system in which inhibition of any one regulatory component (by cell cycle progression or DNA damage for instance) can initiate cohesion generation. This complex regulation reflects the depths to which evolutionary forces are capable of selecting, magnified by the importance of properly segregating the genetic material from which all the cell cycle machinery arise.