GENETICS OF CELL CYCLE & DNA DAMAGE REGULATION IN YEAST

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。存在能够识别和纠正在这些过程中发生的错误的监管网络。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使用MEC、RAD、DPB、DRC和DUN基因在酿酒酵母中进行探测。DPB11和DRc1在dna聚合酶epsilon通路中共同作用，控制dna合成和S阶段检查点。我们将探索这些基因的DNA复制和检查点功能之间的关系，以确定它是如何感知复制问题的。我们将使用遗传和生化分析来确定哪些其他蛋白质与这些蛋白质合作来传递细胞周期停滞信号。当DNA复制被阻断时，细胞周期受阻依赖于RAD53蛋白激酶和两个新的下游基因ESH1和ESH2。我们建议确定Rad53的激活机制，并产生结构性活性突变体来鉴定下游功能。我们将进行ESH1和ESH2的遗传和生化分析，试图确定它们在阻止复制时防止纺锤体延长的作用。我们将在这个检查站寻找更重要的基因。Dun1和Chk1激酶共同作用，防止DNA损伤的后期进入。Dun1被认为通过抑制APC发挥作用，Chk1被认为通过磷酸化Pds1发挥功能，Pds1是一种后期抑制物。我们建议确定它们在这一检查点中的角色，包括生化和遗传分析。我们感兴趣的是Dun1在APC中的靶点以及Pds1磷酸化的意义。DNA损伤诱导基因的转录激活是对DNA损伤的中心反应。我们将使用微阵列分析确定酵母中损伤诱导基因的整个谱系，并确定检查点基因在这些转录本的调控中的作用，以确定是否存在层调控。拟议的研究将有助于我们理解细胞周期控制的机制，这是维持基因组稳定所必需的，从而防止导致癌症的突变积累。

The eukaryotic cell cycle is a cascade of highly complex processes that must occur with striking temporal and spatial precision. There exists regulatory networks capable of recognizing and correcting mistakes occurring during these processes. 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 S. cerevisiae using the MEC, RAD, DPB, DRC, and DUN genes. DPB11 and DRC1 work together in the DNA polymerase epsilon pathway to control DNA synthesis and the S phase checkpoint. We will probe the relationship between the DNA replication and checkpoint functions of these genes to determine how it is sensing replication problems. We will determine what other proteins cooperate with these proteins to transmit cell cycle arrest signals using genetic and biochemical analysis. Cell cycle arrest when DNA replication is blocked depends upon the RAD53 protein kinase and 2 novel downstream genes ESH1 and ESH2. We propose to determine the mechanism of activation of Rad53 and to generate constitutively active mutants to identify downstream function. We will perform genetic and biochemical analyses of ESH1 and ESH2 to try to determine their role in preventing spindle elongation while replication is blocked. We will search for more essential genes in this checkpoint. The Dun1 and Chk1 kinase work together to prevent anaphase entry in response to DNA damage. Dun1 is thought to function by inhibiting the APC and Chk1 is thought to function by phosphorylating Pds1, an anaphase inhibitor. We propose to determine their roles in this checkpoint both biochemical and genetic analyses. We are interested in the target of Dun1 in the APC and the significance of Pds1 phosphorylation. Transcriptional activation of DNA damage inducible genes is a central response to DNA damage. We will determine the entire repertoire of damage-inducible genes in yeast using microarray analysis and the roles of checkpoint genes in the regulation of these transcripts to determine if there are layers of regulation. The proposed studies will contribute to our understanding of the mechanism of cell cycle control that is essential to the maintenance of genomic stability which prevents the accumulation of mutations leading to cancer.