The eukaryotic cell cycle is a cascade of highly complex processes that must occur with striking temporal and spatial precision. This degree of complexity necessitates the existence of a regulatory network capable not only of coordinating these events, but also recognizing and correcting mistakes occurring during these complex processes. For example cells respond to DNA damage and replication blocks in two ways: They arrest the cell cycle to allow time for repair and they induce the transcription of genes facilitating repair. The biochemical pathways ensuring this coordination are called checkpoints. The infrastructure of these checkpoint circuits will be probed in Saccharomyces cerevisiae. Mec 1 and Rad53 are protein kinases essential for arresting the cell cycle and activating repair in response to DNA replication blocks and damage. We propose to explore their function by identifying targets of their pathway by genetic screens. In addition we will isolate potential direct substrates by immunoprecipitation with antibodies that recognize the phosphorylation sites of Mec 1. Mrc 1 is a mediator of the DNA replication response that has a function in DNA replication and is required for Rad53 activation when replication is blocked. Mrc1 is loaded onto chromatin during S phase and phosphorylated in a Mec1-dependent manner in response to stress. We will determine the significance of the phosphorylation and chromatin loading of Mrc 1 and its role in regulation of Rad53. The proper regulation of spindle/kinetochore interactions is important for survival during S phase arrest in S. cerevisiae and requires the DASH complex. We propose to investigate the role of DASH in regulating spindle-kinetochore interactions using biochemical and genetic means. We have also performed genetic analyses that links the DASH complex to ras signaling and we will investigate the role of ras in DASH function. The DNA damage checkpoint inhibits mitotic exit through the Dun1 kinase and Bfa1. We will investigate the role of Bfa1 phosphorylation in maintaining a block to mitotic exit. In addition, we have identified a novel negative regulator of mitotic exit, Amn1. We propose to study the role of Amn1 in allowing cells to turn off the mitotic exit part of the cell cycle to return to G1.
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