CELL CYCLE CHECKPOINT CONTROL IN RESPONSE TO DNA DAMAGE

该项目的长期目标是确定当细胞DNA受损时，细胞如何调节细胞周期的进程。人们早就知道，当基因组的完整性受到损害时，真核细胞会延迟细胞周期的进展。在过去的十年中，人们已经清楚地认识到，基因定义的信号转导途径，即细胞周期检查点，将DNA损伤的检测与细胞周期进程的控制结合起来。受损DNA模板的复制或受损染色体的分离可能对基因组的完整性造成灾难性的后果。因此，这些检查点机制在维持基因组稳定性方面起着至关重要的作用，当受到损害时，可能导致癌症的发生或细胞死亡。人类检查点通路基因ATM的突变导致遗传性疾病共济失调毛细血管扩张症，其特征是进行性神经变性和癌症的高发病率。分裂酵母，Schizosaccharomyces pombe，已被证明是识别细胞周期调节机制和DNA损伤检查点途径组成部分的杰出模型系统。Chk1蛋白激酶首先在裂变酵母中被发现，当DNA被破坏时，它是细胞周期阻滞所必需的。Chkl的同源物已在多种真核生物中被发现，包括青蛙、苍蝇、蠕虫和人类。此外，ATM基因的一个& pombe同源物rad3已被证明在与Chk1相同的途径上起作用。为了理解Chk1在DNA损伤检查点通路中所起的作用，将采用几种方法，利用& pombe可以轻松进行遗传和生化分析。与Chk1相互作用或影响Chk1活性的蛋白质将在利用chkl基因的新型检查点缺陷等位基因的遗传筛选中被鉴定出来。Chk1和Rad24（另一种在DNA损伤检查点通路中发挥作用的蛋白质）之间先前确定的相互作用将被详细描述。Chkl亚细胞定位的决定因素将被确定，因为Chk1在细胞中的位置对Chk1信号的性质以及假定的Chk1靶标的性质都具有重要意义。通过鉴定DNA损伤诱导的磷酸化位点，分析这些位点对Chkl功能的重要性，分析Chk1翻译后磷酸化修饰的要求

The long-term goal of this project is to determine how cells regulate progression through the cell cycle when their DNA has been damaged. It has long been known that eukaryotic cells will delay progression through the cell cycle when the integrity of the genome has been compromised. In the last decade it has become clear that genetically defined signal transduction pathways, known as cell cycle checkpoints, couple the detection of DNA damage to control of cell cycle progression. The replication of damaged DNA templates or the segregation of damaged chromosomes can have catastrophic consequences for the integrity of the genome. Therefore, these checkpoint mechanisms play essential roles in maintaining genomic stability and, when compromised, can contribute to the onset of cancer or to cell death. Mutations in the human checkpoint pathway gene ATM, lead to the genetic disorder Ataxia Telangiectasia which is characterized by progressive neurodegeneration and a high incidence of cancer. The fission yeast, Schizosaccharomyces pombe, has proven to be an outstanding model system for identifying components of the cell cycle regulatory machinery as well as the DNA damage checkpoint pathway. The protein kinase Chk1, first identified in fission yeast, is required for cell cycle arrest when DNA is damaged. Homologues of Chkl have been identified in a variety of eukaryotic organisms including frogs, flies, worms and humans. In addition, an & pombe homologue of the ATM gene, rad3, has been shown to function on the same pathway as Chk1. To understand the role played by Chk1 in the DNA damage checkpoint pathway, several approaches will be taken that capitalize on the ease with which genetic and biochemical analyses can be carried out with & pombe. Proteins that interact with Chk1 or which influence the activity of Chk1 will be identified in genetic screens that make use of novel checkpoint defective alleles of the chkl gene. The previously identified interaction between Chk1 and Rad24, another protein shown to play a role on the DNA damage checkpoint pathway, will be characterized in detail. Determinants of the subcellular localization of Chkl will be identified as the location of Chk1 in the cell has important implications both for the nature of signaling to Chk1 as well as for the nature of putative Chk1 targets. The requirement of posttranslational modification of Chk1 by phosphorylation will be analyzed by identifying the sites of phosphorylation induced by DNA damage and analyzing the importance of those sites for Chkl function