Genetics of Cell Cycle & DNA Damage Regulation in Yeast

描述(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。这种程度的复杂性要求存在一个监管网络，不仅能够协调这些事件，而且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损伤和复制障碍做出反应：它们阻止细胞周期，以便有时间进行修复；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被称为检查点。这些检查点电路的基础结构将在酿酒酵母中进行探索。MEC 1和Rad53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来识别它们途径的靶点，以探索它们的功能。此外，我们将通过识别Mec 1磷酸化位点的抗体的免疫沉淀来分离潜在的直接底物。mRC 1是DNA复制反应的中介，在DNA复制中具有功能，当复制被阻止时，Rad53的激活是必需的。在S期，mRc1被加载到染色质上，并以依赖于mec1的方式磷酸化以响应压力。我们将确定MRC1的磷酸化和染色质负载的意义及其在调控Rad53中的作用。在酿酒酵母中，纺锤体/着丝粒相互作用的适当调节对于酿酒酵母S相停滞期间的生存是重要的，并且需要DASH复合体。我们建议利用生物化学和遗传学手段研究DASH在调节纺锤体-着丝粒相互作用中的作用。我们还进行了将DASH复合体与ras信号联系起来的遗传分析，并将研究ras在DASH功能中的作用。DNA损伤检查点通过Dun1激酶和Bfa1抑制有丝分裂退出。我们将研究Bfa1的磷酸化在维持对有丝分裂退出的阻断中的作用。此外，我们还发现了一个新的有丝分裂退出负调控因子Amn1。我们建议研究Amn1在允许细胞关闭有丝分裂退出部分细胞周期返回G1期中的作用。

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.