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

裂殖酵母 DNA 损伤反应的新决定因素

基本信息

批准号：
7982831
负责人：
MATTHEW J O'CONNELL
金额：
$ 31.79万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-08-20 至 2014-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7982831
关键词：
Age Aging Alleles Antineoplastic Agents Biochemical Biological Biological Models Biology Cancer Etiology Cell Aging Cell Cycle Cell Cycle Checkpoint Cell Cycle Progression Cell Cycle Regulation Cells Checkpoint kinase 1 DNA DNA Damage DNA Double Strand Break DNA Repair DNA Repair Pathway DNA biosynthesis DNA damage checkpoint Data Defect Dependence Development Drosophila genus Drug Delivery Systems Enzyme Activation Enzymes Event Exonuclease Family Fission Yeast Flap Endonucleases G2 Phase Generations Genes Genetic Genetic Recombination Genome Genome Components Genomic Instability Genomics Goals Homologous Gene Human Invaded Laboratories Learning Lesion Malignant Neoplasms Mediating Minor Planets Mitosis Mitotic Modeling Monitor Pathway interactions Phase Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Plant Roots Primary Lesion Process Proliferating Protein Dephosphorylation Proteins Quality Control Reagent Recruitment Activity Regulation SS DNA BP Series Signal Pathway Signal Transduction Single-Stranded DNA Specificity Superhelical DNA Time Type I DNA Topoisomerases Yeasts checkpoint kinase 2 environmental mutagens functional restoration gene function genetic analysis genetic manipulation helicase human disease in vivo mutant novel nuclease overexpression prevent public health relevance recombinational repair repaired research study response senescence

项目摘要

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.

描述（由申请人提供）：该提案集中于细胞对DNA损伤的反应。实验是在裂变酵母裂菌酵母POMBE中进行的，该模型系统提供了细胞周期和检查点控制的范式。 S. Pombe具有监测基因组完整性的两个主要检查点响应。这些是S阶段检查点和DNA损伤检查点。当DNA复制的进展受到阻碍时，激活了S期检查点，尽管其效应激酶CDS1，但持续停滞的复制叉的稳定性仍保持稳定，以促进DNA复制的恢复。所有形式的DNA损伤都激活了DNA损伤检查点，并通过其效应激酶CHK1损伤，将细胞周期停止，以便可以在进入有丝分裂之前修复病变。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癌症标志的基因组不稳定性至关重要。检查点对于细胞衰老和衰老的生物学以及反应环境诱变剂也至关重要。因此，对其生物学的详细理解是人类疾病的核心。关于这些检查站的协调方式，如何启动以及如何终止，存在着出色的问题。在当前对检查点信号通路的理解中，确定这些检查点的所有组成部分并填补孔的所有组件是我们实验室和该项目的长期目标。我们介绍了大量的初步数据，其中已经发现了检查点信号的新方面。首先，我们已经开始确定CHK1激活和失活的机制，这是基本生物学和靶向CHK1的抗癌药物的重要问题。其次，我们确定了新的调节机制，通过这些机制，细胞在块中促进DNA损伤至DNA复制。这些是通过新的检查点介导的对复制叉处的拓扑异构酶I分子的调节。此外，我们已经确定了两个与检查点信号的启动事件有关的其他新检查点基因。这两个基因过表达时，都将功能恢复为CHK1的肌电等位基因，并且似乎正在放大启动检查点信号。这些基因的无效等位基因表明它们是DNA损伤检查点所需的。它们似乎在检查点途径的最上游端起作用，其中包括检查点控件的最低知识方面。为了进一步进一步研究，我们提出了利用遗传，生化和细胞生物学方法的三个目标。首先，我们使用为S. pombe酶开发的一系列独特的试剂来剖析CHK1激活和灭活的机制。其次，我们剖析了拓扑异构酶I介导的检查点激活，以及该酶如何受S-相检查点调节。最后，我们表征了一个相关的核酸酶家族，这些核酸酶似乎正在将原发性病变处理为激活单链DNA的检查点。鉴于检查点基因函数的高保守性，这些新的检查点成分在人类细胞中的起作用可能相似。公共卫生相关性：细胞以显着的保真度增殖，质量控制的缺陷（称为检查点）是癌症，衰老和对环境诱变剂的反应的根本原因。由于这些过程的起源很古老，因此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。