Genetics of Cell Cycle & DNA Damage Regulation in Yeast

描述（由申请人提供）：真核细胞周期是一系列高度复杂的过程，必须以惊人的时间和空间精度发生。这种程度的复杂性需要一个监管网络的存在，不仅能够协调这些事件，而且还能够识别和纠正在这些复杂过程中发生的错误。例如，细胞以两种方式对DNA损伤和复制阻断做出反应：它们阻止细胞周期以留出时间进行修复，并且它们诱导促进修复的基因的转录。确保这种协调的生化途径称为检查点。这些检查点电路的基础设施将在酿酒酵母中进行探索。Mec 1和Rad 53是阻止细胞周期和激活修复以响应DNA复制阻断和损伤所必需的蛋白激酶。我们建议通过基因筛选确定其途径的靶点来探索其功能。此外，我们将分离潜在的直接底物免疫沉淀抗体识别磷酸化位点的Mec 1。MRC 1是DNA复制应答的介体，其在DNA复制中具有功能，并且在复制被阻断时是Rad 53活化所需的。Mrc 1在S期被加载到染色质上，并以Mec 1依赖的方式磷酸化以响应压力。我们将确定MRC 1的磷酸化和染色质负载的意义及其在调节Rad 53中的作用。纺锤体/动粒相互作用的适当调节对于S期停滞期的存活是重要的。酿酒酵母和需要DASH复合物。我们建议使用生物化学和遗传学手段研究DASH在调节纺锤体-动粒相互作用中的作用。我们还进行了将DASH复合物与ras信号转导联系起来的遗传分析，我们将研究ras在DASH功能中的作用。DNA损伤检查点通过Dun 1激酶和Bfa 1抑制有丝分裂退出。我们将研究Bfa 1磷酸化在维持有丝分裂出口阻滞中的作用。此外，我们已经确定了一种新的负调节有丝分裂出口，Amn 1。我们建议研究Amn 1在允许细胞关闭细胞周期的有丝分裂出口部分以返回G1中的作用。

DESCRIPTION (provided by applicant): 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.