New determinants of the DNA damage response in the fission yeast S. pombe

描述(由申请人提供)：本提案以细胞对DNA损伤的反应为中心。这些实验是在裂解酵母裂殖酵母中进行的，裂殖酵母是一个提供了细胞周期和检查点控制范例的模型系统。S.pombe有两个主要的检查点反应来监测基因组的完整性。这两个是S期内检查站和DNA损伤检查站。当DNA复制受阻时，S期内检查点被激活，尽管其效应蛋白激酶CDS1，但保持停滞的复制叉的稳定性，以促进DNA复制的恢复。所有形式的DNA损伤都会激活DNA损伤检查点，并通过其效应蛋白激酶Chk1停止细胞周期，以便在进入有丝分裂之前修复损伤。这些检查点是基因组完整性的关键决定因素，因此它们的功能对于防止基因组不稳定至关重要，基因组不稳定是癌症的一个标志。检查点在细胞老化和衰老的生物学以及对环境诱变剂的反应中也是至关重要的。因此，对它们生物学的详细了解是人类疾病的核心。关于如何协调这些检查站、如何启动检查站以及如何终止检查站，还有一些悬而未决的问题。我们实验室和这个项目的长期目标是识别这些检查点的所有组件，并填补目前对检查点信号通路的理解中的漏洞。我们提供了大量的初步数据，在这些数据中，检查点信号的新方面已经被发现。首先，我们已经开始确定Chk1的激活和失活机制，这在基础生物学和针对Chk1的抗癌药物的开发中都是一个重要的问题。其次，我们确定了新的调控机制，通过这些机制，细胞可以在阻止DNA复制的过程中防止DNA损伤。这些都是通过新的检查点介导的拓扑异构酶I分子在复制叉处的调节。此外，我们还鉴定了另外两个新的检查点基因，它们与检查点信号的启动事件有关。当过度表达时，这两个基因将功能恢复到Chk1的亚型等位基因，并似乎放大了启动检查点的信号。这些基因的零等位基因表明它们是DNA损伤检查点所必需的。它们似乎在检查点路径的最上游端发挥作用，其中包括对检查点控制最不了解的方面。为了进一步进行这些初步研究，我们提出了利用遗传学、生化和细胞生物学方法的三个目标。首先，我们使用我们为S.pombe酶开发的一系列独特的试剂剖析了Chk1激活和失活的机制。其次，我们剖析了拓扑异构酶I介导的检查点激活，以及该酶是如何被S期内的检查点调控的。最后，我们描述了一系列相关的核酸酶，这些核酸酶似乎正在将原发病变处理为激活单链DNA的检查点。鉴于检查点基因功能的高度保守性，这些新的检查点组件很可能在人类细胞中发挥类似的功能。 与公共卫生相关：细胞以惊人的保真度增殖，质量控制中的缺陷，即所谓的检查点，是癌症、衰老和对环境诱变剂反应的根本原因。因为这些过程由来已久，我们使用简单的酵母细胞来识别与人类疾病相关的新基因。我们的建议分析了细胞对DNA损伤的反应中的三个新基因。

DESCRIPTION (provided by applicant): This proposal centers on the cellular response to DNA damage. The experiments are performed in the fission yeast Schizosaccharomyces pombe, a model system that has provided a paradigm of cell cycle and checkpoint control. S. pombe has two major checkpoint responses that monitor genome integrity. These are the intra-S-phase checkpoint and the DNA damage checkpoint. The intra-S-phase checkpoint is activated when the progression of DNA replication is impeded, and though its effector kinase Cds1, the stability of stalled replication forks is maintained to facilitate resumption of DNA replication. All forms of DNA damage activate the DNA damage checkpoint, and through its effector kinase Chk1, the cell cycle is halted such that the lesion can be repaired prior to entry into mitosis. These checkpoints are key determinants of genome integrity, and as such their function is crucial to prevent the genome instability that is a hallmark of cancer. Checkpoints are also crucial in the biology of cellular ageing and senescence, and in the response environmental mutagens. Thus, a detailed understanding of their biology is central to human disease. There are outstanding issues as to how these checkpoints are coordinated, how they are initiated and how they are terminated. It is the long-term goal of our laboratory, and of this project, to identify all components of these checkpoints and fill holes in the current understanding of the checkpoint signaling pathways. We present a significant body of preliminary data in which novel aspects of checkpoint signaling have been uncovered. First, we have begun to determine mechanisms of activation and inactivation of Chk1, an important issue in both basic biology and in the development of anti-cancer drugs that target Chk1. Second, we identify new regulatory mechanisms through which cells prevent DNA damage during blocks to DNA replication. These are via novel checkpoint-mediated regulation of Topoisomerase I molecules at the replication forks. Further, we have identified two additional new checkpoint genes that are implicated in the initiating events of checkpoint signaling. Both genes, when overexpressed, restore function to a hypomorphic allele of chk1, and appear to be amplifying the initiating checkpoint signal. Null alleles of these genes show they are required for the DNA damage checkpoint. They appear to function at the most upstream end of the checkpoint pathway, which include the least understood aspects of checkpoint control. To further these preliminary studies, we propose three aims utilizing genetic, biochemical and cell biological approaches. First, we dissect mechanisms of Chk1 activation and inactivation using a unique series of reagents we have developed for the S. pombe enzyme. Second, we dissect Topoisomerase I mediated checkpoint activation, and how this enzyme is regulated by the intra-S- phase checkpoint. Finally, we characterize a family of related nucleases that appear to be processing primary lesions into checkpoint activating single-stranded DNA. Given the high conservation of checkpoint gene function, these new checkpoint components are likely to function similarly in human cells. PUBLIC HEALTH RELEVANCE: Cells proliferate with remarkable fidelity, and defects in quality controls, known as checkpoints, are at the root cause of cancer, aging and responses to environmental mutagens. Because these processes are ancient in origin, we use simple yeast cells to identify new genes that are relevant to human disease. Our proposal analyses three new genes in the response of cells to DNA damage.