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

描述(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基因变化而产生的。这些变化中的许多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。其中最核心的是S阶段的DNA损伤检查点，这是一种减缓复制以应对DNA损伤的机制。该检查点有两个分支：一个调节复制起点的激活，另一个需要MRN(Mre11-Rad50-Nbs1)重组复合体。MRN依赖的分支的机制尚不清楚。然而，在人类和小鼠中的遗传证据表明，S期DNA损伤检查点的这一分支对于预防癌症至关重要；携带mre11或NBS1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是必不可少的，并将从根本上影响对该检查点的后续研究。拟议的实验旨在测试MRN依赖的分支诱导复制耦合重组的假设。这一假设将通过量化复制过程中DNA损伤引起的重组速度，并通过确定这种重组是否以及如何受到检查点的调控来直接检验。对MRN蛋白的互补生化和遗传分析将确定它们在检查点中的特定机制角色，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庞贝裂殖酵母作为模型系统。分裂酵母和人类之间检查点的保守使分裂酵母成为研究这些重要的DNA损伤监测途径的一个很好的模型。可用于裂解酵母的强大的遗传和生化工具使其能够快速识别关键途径成员，并严格测试关于其功能的假说。了解裂解酵母S期的DNA损伤检查点将为理解人类检查点如何维持基因组稳定提供重要的框架。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.