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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）：该提案集中于对DNA损伤的细胞反应。实验在裂殖酵母裂殖酵母中进行，裂殖酵母是一种模型系统，提供了细胞周期和检查点控制的范例。S.粟酒裂殖酵母有两个主要的检测点反应，监测基因组的完整性。这些是S期内检查点和DNA损伤检查点。当DNA复制的进程受阻时，S期内检查点被激活，并且通过其效应激酶Cds1，维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，并通过其效应激酶Chk1停止细胞周期，从而使损伤在进入有丝分裂之前得到修复。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作为癌症标志的基因组不稳定性至关重要。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至关重要。因此，对它们生物学的详细了解对人类疾病至关重要。在这些检查站如何协调、如何设立和如何终止方面存在着一些悬而未决的问题。我们实验室和本项目的长期目标是确定这些检查点的所有组成部分，并填补目前对检查点信号通路的理解中的漏洞。我们提供了大量初步数据，其中发现了检查点信号的新方面。首先，我们已经开始确定Chk1的激活和失活机制，这是基础生物学和靶向Chk1的抗癌药物开发中的一个重要问题。其次，我们确定了新的调控机制，通过这些机制，细胞在阻止DNA复制时防止DNA损伤。这些是通过拓扑异构酶I分子在复制叉处的新检查点介导的调节。此外，我们已经确定了另外两个新的检查点基因，它们与检查点信号传导的起始事件有关。这两个基因，当过度表达时，恢复功能的chk1的亚型等位基因，并出现放大启动检查点信号。这些基因的等位基因表明它们是DNA损伤检查点所必需的。它们似乎在检查点通路的最上游端起作用，其中包括检查点控制的最不了解的方面。为了进一步这些初步研究，我们提出了三个目标，利用遗传，生物化学和细胞生物学的方法。首先，我们使用我们为S.粟酒酶其次，我们剖析了拓扑异构酶I介导的检查点激活，以及这种酶如何受到S期内检查点的调节。最后，我们描述了一个家族的相关核酸酶，似乎是加工成检查点激活单链DNA的原发性病变。鉴于检查点基因功能的高度保守性，这些新的检查点组件可能在人类细胞中具有类似的功能。公共卫生相关性：细胞以惊人的保真度增殖，而质量控制的缺陷，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。由于这些过程起源古老，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。我们的提案分析了细胞对DNA损伤的反应中的三个新基因。