The Role of MRN in the S-Phase DNA Damage Checkpoint

描述（由申请人提供）：人类癌症是通过一系列将正常细胞转化为恶性肿瘤的遗传变化而产生的。许多这些变化是由基因组重排和复制过程中的其他错误引起的。为了防止和修复这种复制错误，细胞进化出了DNA损伤检查点，这是一套复杂的DNA质量控制机制。其中的核心是S期DNA损伤检查点，这是一种减缓DNA损伤复制的机制。这个检查点有两个分支：一个是调节复制起点的激活，另一个需要MRN（Mre 11-Rad 50-Nbs 1）重组复合物。MRN依赖性分支的机制尚不清楚。然而，人类和小鼠的遗传学证据表明，S期DNA损伤检查点的这个分支对预防癌症至关重要; Mre 11或Nbs 1突变的人类患者容易发生各种早发性恶性肿瘤。了解这一检查点的机制对于了解这些癌症的病因至关重要，并将从根本上影响随后对这一检查点的研究。所提出的实验被设计为检验MRN依赖性分支用于诱导复制偶联重组的假设。通过定量复制过程中DNA损伤诱导的重组率，并确定该重组是否以及如何受检查点调控，将直接检验该假设。对MRN蛋白的补充生化和遗传分析将确定它们在检查点中的特定机制作用，并将为研究一般检查点机制提供工具。这些实验将利用裂殖酵母粟酒裂殖酵母作为模型系统。裂变酵母和人类之间检查点的保守性使得裂变酵母成为研究这些重要DNA损伤监测途径的极好模型。裂变酵母强大的遗传和生物化学工具使其能够快速识别关键途径成员并严格测试有关其功能的假设。了解裂变酵母S期DNA损伤检查点将为理解人类检查点如何维持基因组稳定性提供重要框架。这一认识将为人类癌症的治疗和预防带来新的治疗靶点和诊断工具。

DESCRIPTION (provided by applicant): Human cancers arise through a series of genetic changes that transform normal cells into malignant tumors. Many of these changes are caused by genomic rearrangements and other errors during replication. To prevent and repair such replication errors, cells have evolved DNA damage checkpoints, a sophisticated set of DNA quality control mechanisms. Central among them is the S-phase DNA damage checkpoint, a mechanism that slows replication in response to DNA damage. This checkpoint has two branches: one that regulates the activation of replication origins, and one that requires the MRN (Mre11-Rad50-Nbs1) recombination complex. The mechanism of the MRN-dependent branch is unknown. However, genetic evidence in humans and mice suggest that this branch of the S-phase DNA damage checkpoint is crucial for preventing cancer; human patients with mutations in Mre11 or Nbs1 are prone to a variety of early-onset malignancies. Understanding the mechanism of this checkpoint is essential for understanding the etiology of these cancers and will fundamentally affect the way subsequent studies of this checkpoint are approached. The proposed experiments are designed to test the hypothesis that the MRN-dependent branch acts to induce replication-coupled recombination. This hypothesis will be directly tested by quantitating the rate of recombination induced by DNA damage during replication and by determining if, and how, this recombination is regulated by the checkpoint. Complementary biochemical and genetic analysis of the MRN proteins will identify their specific mechanistic roles in the checkpoint, and will provide tools for studying the general checkpoint mechanism. These experiments will take advantage of the fission yeast Schizosaccharomyces pombe as a model system. The conservation of checkpoints between fission yeast and humans makes fission yeast an excellent model for investigating these vital DNA damage surveillance pathways. The powerful genetic and biochemical tools available for fission yeast make it possible to rapidly identify key pathway members and rigorously test hypotheses about their functions. Understanding the fission yeast S-phase DNA damage checkpoint will provide an important framework for understanding how the human checkpoint maintains genomic stability. This understanding will lead to new therapeutic targets and diagnostic tools for the treatment and prevention of human cancer.