Chromosome cohesion and condensation in Saccharomyces cerevisiae.

University of Minnesota Ph.D. dissertation. August 2011. Major: Molecular, Cellular, Developmental Biology and Genetics. Advisor: Duncan J Clarke, PhD. 1 computer file (PDF); vii, 132 pages, appendix p. 100-132.Sister chromatid cohesion and chromosome condensation are two essential cell cycle processes for maintaining genome stability. Pds1 is the only known regulator for maintaining cohesion between sister chromatids during S phase till anaphase onset by inhibiting Esp1 activity, which is important for preventing aberrant chromosome segregation. However, pds1 null yeast are viable and able to keep cohesion during S phase. This indicates a redundant pathway maintains sister chromatid cohesion in the absence of Pds1. We have identified the Pds1-independent mechanism involved in S phase sister chromatid cohesion. This mechanism requires the function of two B-type cyclins, Clb5 and Clb6, as well as Cdc28. When DNA replication is efficient, either the Pds1- or Clb5/Clb6-dependent mechanism is sufficient to maintain sister chromatid cohesion. However, both mechanisms are required for S phase cohesion under conditions of replication stress. Further investigation revealed that cells lacking Clb5 and Clb6 have reduced levels of chromatin associated cohesin under conditions of replication stress. Further, this cohesion defect requires spindle tension to be observed by examining TRP1 locus separation. In conclusion, yeast cells maintain sister chromatid cohesion by a Clb5/Clb6 dependent mechanism for efficiently loading cohesin onto chromatin during S phase as well as a Pds1 dependent mechanism for inhibiting the protease activity of Esp1 before anaphase onset. Chromosome condensation requires the function of the condesin complex. Recent studies mainly focused on the ATPase activity of condensin in introducing DNA supercoiling. We studied how condensin is regulated through the cell cycle. We found that the protein levels of condensin subunits are cell cycle regulated. Chromosomes condense in response to higher condensin protein abundance. Chromosome decondensation at the end of the cell cycle requires Smc4 proteolysis mediated by APC/C ubiquitin ligase. This Smc4 protein turnover needs the function of Mad2 in the absence of nocodazole, but Mad2 is dispensable for Smc4 proteolysis under conditions of pre-anaphase arrest in the presence of nocodazole. In addition to protein abundance, phospho modification of Smc4 by Cdc28 also regulates the timing of chromosome condensation. This phospho modification of Smc4 destabilized Smc4 protein as well as preventing premature chromosome condensation early in the cell cycle. Collectively, we studied the underlying mechanisms of two essential events for genome stability: sister chromatid cohesion and chromosome condensation. Both events are under regulation by multiple mechanisms to ensure the faithfulness. Understanding these mechanisms using budding yeast would avail against the human diseases caused by genome instability