Checkpoint Regulation of Stalled and Collapsed Replication Forks

停滞和崩溃复制叉的检查点调节

• 批准号: 8396111
• 负责人: Frank Benjamin Couch
• 金额: $ 2.69万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8396111
• 关键词: ATR protein kinase; Affect; Ataxia-Telangiectasia-Mutated protein kinase; Base Pairing; Biochemical; Bromodeoxyuridine; CHES1 gene; Camptothecin; Cell Cycle Progression; Cells; Cosmic Radiation; Cytometry; DNA; DNA Damage; DNA Structure; DNA biosynthesis; Data; Development; Double Strand Break Repair; Event; Excision; Fiber; Figs - dietary; Generations; Genes; Genetic; Genetic Materials; Genome; Genomics; Histones; Human; Immunofluorescence Immunologic; Label; Lead; Lesion; Life; Literature; Malignant Neoplasms; Mammalian Cell; Mass Spectrum Analysis; Mediating; Metabolism; Methodology; Mutation; Nucleotides; Outcome; Pathway interactions; Phosphorylation; Phosphotransferases; Process; Protein Binding; Protein Kinase; Proteins; Reagent; Recovery; Regulation; Reporting; Research; Resolution; Signal Transduction; Single-Stranded DNA; Site; Source; Staining method; Stains; Streptavidin; Stress; Stress Response Signaling; Stretching; Structure; Techniques; Technology; Testing; Ultraviolet Rays; Variant; Work; Yeasts; assault; ataxia telangiectasia mutated protein; base; biological adaptation to stress; cancer cell; chromatin immunoprecipitation; hydroxyurea; inhibitor/antagonist; killings; novel; nuclease; nucleoside analog; prevent; repaired; response

DESCRIPTION (provided by applicant): The DNA damage response (DDR) senses and repairs genotoxic insults, arising from both endogenous metabolic processes and exogenous sources such as ultraviolet or cosmic radiation. Misrepair of these lesions results in mutations which lead to the development of cancer. Template damage, nucleotide imbalances, and difficult to replicate sequences cause replication stress and provide another potential source of mutations. The Replication Stress Response (RSR), an intrinsic cellular response to replication stress, functions during normal and stressed replication to stabilize stalled replication forks and promote the completion of DNA replication. The protein kinase ATR senses and responds to stalled replication forks, where it phosphorylates numerous substrates to begin RSR signaling. This proposal will use hydroxyurea (HU), which effects rapid replication fork stalling, to study th function of ATR using newly developed inhibitors. I hypothesize that ATR regulates stalled and collapsed replication forks to promote fork restart and avoid pathogenic fork intermediates. To test this hypothesis, I will utilize the newly developed iPOND methodology to probe replisome stability in response to HU and ATR inhibition. Furthermore, I will determine the involvement of several genetic pathways in modulating the severe HU sensitivity of ATR inhibited cells and in the generation of an unusual DNA structure where the newly synthesized DNA becomes single stranded. In this analysis, I will include the structure specific nuclease MUS81, which my preliminary data suggests functions upstream of nascent-strand ssDNA generation. Previous reports indicate that MUS81-deficient cells suffer from HU sensitivity, so this proposal will test the concept that checkpoint signaling regulates MUS81 activity at stalled forks. Finally, I will combine the iPOND methodology with the unbiased approach, mass spectrometry, to identify new RSR proteins. The recent literature includes several reports of new RSR factors. Given this rate of discovery, many new RSR factors likely remain undiscovered. I anticipate that this powerful approach will allow identification of new RSR proteins associated with newly synthesized DNA under normal, stressed, and ATR inhibited conditions. In summary, this proposal will provide a significant step forward in our understanding of how ATR functions in response to replication stress to mediate protein recruitment, replisome stabilization, and restart of replication. PUBLIC HEALTH RELEVANCE: The process of DNA replication provides a significant challenge to genome integrity, as template damage, nucleotide imbalances, and difficult to replicate sequences cause replication stress. Checkpoint signaling responds to this stress to prevent cell cycle progression in the presence of damage and coordinate repair of damaged replication forks. This proposal combines novel inhibitors of the ATR checkpoint kinase with state-of- the-art technologies such as iPOND and DNA fiber labeling to elucidate the mechanism by which the checkpoint stabilizes stalled replication forks and protects genome integrity.

描述（由申请人提供）：DNA损伤反应（DDR）感觉和修复遗传毒性损伤，这是由内源性代谢过程和外源性来源（例如紫外线或宇宙辐射）引起的。这些病变的误解导致突变导致癌症的发展。模板损伤，核苷酸失衡和难以复制序列会导致复制应力并提供另一个潜在的突变来源。复制应力反应（RSR），一种对复制应力的固有细胞反应，在正常和应力复制过程中的功能，以稳定停滞的复制叉和 促进DNA复制的完成。蛋白激酶ATR感应并响应停滞的复制叉，在那里它磷酸化了许多底物以开始RSR信号传导。该提案将使用羟基脲（HU），影响快速复制的叉档，以使用新开发的抑制剂来研究ATR的TH功能。我假设ATR调节停滞不前的复制叉以促进叉子重新启动并避免致病叉中间体。为了检验这一假设，我将利用新开发的IPOND方法来探测对HU和ATR抑制作用的探测。此外，我将确定几种遗传途径在调节ATR抑制细胞的严重HU敏感性以及新合成的DNA不寻常的DNA结构中的严重HU敏感性中的参与。在此分析中，我将包括结构特定的核酸酶MUS81，我的初步数据表明，新生链ssDNA生成的功能。先前的报告表明，MUS81缺陷型细胞患有HU敏感性，因此该提案将测试检查点信号传导调节失速叉子的MUS81活性的概念。最后，我将将IPOND方法与无偏的方法（质谱法）结合在一起，以识别新的RSR蛋白。最近的文献包括有关新RSR因素的几份报告。鉴于这种发现率，许多新的RSR因素可能仍然未被发现。我预计这种强大的方法将允许在正常，压力和ATR抑制条件下与新合成的DNA相关的新RSR蛋白。总而言之，该提案将在我们了解ATR如何响应复制应力方面的功能以介导蛋白质募集，重新稳定和重新启动方面提供重要的一步。 复制。 公共卫生相关性：DNA复制的过程为基因组完整性带来了重大挑战，是模板损伤，核苷酸失衡和难以复制的序列引起复制应力。检查点信号对这种应力做出响应，以防止在损坏存在并坐标损坏的复制叉的维修时进行细胞周期的进程。该建议将ATR检查点激酶的新型抑制剂与最先进的技术（例如IPOND和DNA纤维标记）结合在一起，以阐明检查点可以稳定停滞的复制叉并保护基因组完整性的机制。