The accurate segregation of chromosomes is key to the successful transmission of genetic information to daughter cells. Deficiencies in this process are associated with tumorigenesis, miscarriages, and congenital disorders. The establishment of cohesion between replicated sister chromatids and its maintenance through metaphase are essential prerequisites for the segregation of sister chromatids during mitosis. Cohesion proximal to the kinetochores on paired sister chromatids is especially robust, and is thought to sterically constrain the kinetochores in an orientation that promotes the formation of bipolar microtubule attachments. Evidence is now emerging that the kinetochore also behaves as an enhancer for the recruitment of cohesin, a multisubunit complex of cohesion proteins, over large pericentric domains. The long-term goal of this proposal is to use the Saccharomyces cerevisiae model system to elucidate the molecular mechanism through which the kinetochore coordinates both microtubule binding and cohesion activities and how these activities function to maintain genomic stability. Our focus will be to determine the functional significance of these extended pericentric cohesin domains and the role of kinetochore-microtubule attachments in their generation. The fidelity of chromosome segregation will be examined genetically following the deletion of a pericentric cohesin domain and when the activity of the centromeric enhancer for cohesin binding is limited. Deletions and insertions of potential boundary elements wifi be constructed to determine whether these elements contribute to the creation of distinct pericentric cohesin domains. Standard techniques wifi be used to test whether these regions possess specialized chromatin structures that mediate boundary function, and a yeast one-hybrid screen will be performed to identify protein factors required for boundary activity. A high copy suppressor screen will be performed to identify factors that recruit cohesin to pericentric DNA and novel proteins identified in this screen will be subjected to standard genetic and biochemical analyses to assess in vivo function. The regulation of the centromeric enhancer by kinetochore-microtubule interactions will be examined in mutants with altered microtubule binding activities and the relative contributions of microtubule attachments and the tension that results from these attachments to the regulation of enhancer activity will be determined using a centromere that can form oniy a monopolar spindle microtubule attachment
