Formation of a Neocentromere at a DSB Site

DSB 位点新着丝粒的形成

• 批准号: 9101005
• 负责人: KATSUMI KITAGAWA
• 金额: $ 7.38万
• 依托单位: RESEARCH INST NATIONWIDE CHILDREN'S HOSP
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-15 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9101005
• 关键词: Adverse effects; Apoptotic; Binding; Cell Death; Cell Survival; Cells; Centromere; Chemical Agents; Chromatin; Chromatin Structure; Chromosomal Breaks; Chromosomal Rearrangement; Chromosome Segregation; Chromosomes; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Repair Pathway; DNA damage checkpoint; DNA lesion; DNA replication fork; Double Strand Break Repair; Exposure to; Failure; Foundations; Genomic Instability; Goals; Hela Cells; Histone H3; Immunofluorescence Microscopy; Inherited; Ionizing radiation; Kinetics; Kinetochores; Lead; Maps; Measures; Mediating; Microtubules; Mitosis; Mitotic; Modeling; Molecular; Molecular Target; Mutation; Pathway interactions; Play; Process; Proteins; Radiation Tolerance; Radiation therapy; Recruitment Activity; Regulation; Research Project Grants; Role; Signal Pathway; Site; Structure; Testing; Therapeutic; Time; Variant; Work; base; cancer cell; cancer therapy; centromere protein A; chemotherapeutic agent; genetic information; inhibitor/antagonist; insight; neoplastic cell; novel; protein complex; public health relevance; repaired; research study; response; segregation; tumorigenesis

 DESCRIPTION (provided by applicant): The histone H3 variant, centromere protein A (CENP-A), also localizes to DNA at double-strand breaks (DSBs), implying involvement in responses to DNA damage. CENP-A is normally a constituent of centromere specific chromatin, which forms the foundation for assembly of the kinetochore, a proteinaceous structure comprising a number of conserved protein complexes, that serve as the interface between chromosomes and spindle microtubules. CENP-A plays a crucial role in centromere identity and kinetochore assembly, and is essential to centromere and kinetochore functions, but why it should be recruited to DSB foci is not known. Our working hypothesis is that CENP-A is recruited to DSBs to create a DNA damage-specific form of chromatin to which DNA-damage checkpoint/repair proteins are recruited. Based on this idea, we propose a new model to describe an important mechanism of survival of unrepaired DSBs. In this model, unrepaired DNA damage produces acentric chromosome fragments that may harbor essential genetic information. To stably inherit these acentric fragments, a neocentromere must be formed at the DSB site marked by CENP-A protein on the broken chromosome fragment. The neocentromere facilitates microtubule attachment and proper segregation during mitosis. We hypothesize that incorporation of CENP-A at a DSB site can result in the formation of a neocentromere, and survival of cells following the failure to repair DSBs induced by chemotherapeutic agents or radiation therapy. The specific objective of this proposal is to determine the role of CENP-A in the DDR. Aim 1. Determine significance of CENP-A localization at DSB sites. We will introduce DSBs into chromosomes in cells depleted of CENP-A, then analyze DNA damage checkpoint activity and DNA repair kinetics at the DSB site, and determine if recruitment of known DNA damage checkpoint/repair proteins to DSB sites is abrogated. Furthermore, we will measure DNA repair activity, and survival in cells depleted of CENP-A. Aim 2. Determine if neocentromere formation occurs at the site of DSBs. We hypothesize that a neocentromere or a "pseudo"-neocentromere assembles at the DSB site in a CENP-A-dependent manner. Some spindle assembly checkpoint components have been identified at DSBs, but it is unknown whether a functional kinetochore, which can capture microtubules, is assembled. To test this hypothesis, we will perform immunofluorescence microscopy to detect all the kinetochore components. We will also examine whether neocentromere formation is increased when DDR pathways fail. These studies will provide novel insight into the role of CENP-A in cell survival. The research project will establish a novel role of CENP-A in the DDR, and reshape the map of the DNA-damage signaling pathway. If neocentromere formation occurs at the DSB site, it will represent a previously unknown mechanism for protecting cells from genome instability in response to DNA damage.

 描述（由应用程序提供）：组蛋白H3变体，Centromere蛋白A（CENP-A），也定位在双链断裂（DSB）的DNA（DSB），这意味着参与了对DNA损伤的响应。 CENP-A通常是Centromere特异性染色质的构造，它构成了构成动物学的基础，这是一种完成许多保守蛋白质复合物的蛋白质结构，它是染色体和纺锤体微管之间的界面。 CENP-A在丝粒身份和动力学组装中起着至关重要的作用，对于丝粒和动力学函数至关重要，但是为什么不知道它应该招募到DSB FOCI。我们的工作假设是，将CENP-A募集到DSB中，以创建DNA损伤特异性的染色质形式，将DNA破坏检查点/修复蛋白募集到其中。基于这个想法，我们提出了一个新模型，以描述未修复的DSB生存的重要机制。在此模型中，未修复的DNA损伤会产生可能带有基本遗传信息的敏感染色体片段。为了稳定地继承这些过度的片段，必须在破碎的染色体片段上以CENP-A蛋白为标志的DSB位点形成新中心粒。 Necentromere在有丝分裂过程中寄养了微管的附着和适当的分离。我们假设在DSB部位将CENP-A的结合结合起来会导致新中心粒脑的形成，并且未能修复由化学治疗剂或放射治疗诱导的DSB后细胞的存活。 该提案的具体目标是确定CENP-A在DDR中的作用。目标1。确定CENP-A定位在DSB站点的重要性。我们将在耗尽CENP-A的细胞中将DSB引入DSB，然后分析DSB部位的DNA损伤检查点活性和DNA修复动力学，并确定是否将已知的DNA损伤检查点/修复蛋白募集到DSB位点。此外，我们将测量DNA修复活性，并在CENP-A耗尽的细胞中存活。目标2。确定在DSB的部位是否发生了新中心粒形成。我们假设新自中心粒或“伪” - 非上粒粒子以CENP-A依赖性方式组装在DSB位点。在DSB上已经确定了一些主轴组件检查点组件，但是尚不清楚可以组装的功能性动力学能够捕获微管。为了检验该假设，我们将执行免疫荧光显微镜检测所有动力学成分。我们还将检查当DDR途径失败时，新中心粒脑层的形成是否会增加。这些研究将为CENP-A在细胞存活中的作用提供新的见解。 该研究项目将在DDR中建立CENP-A的新作用，并重塑DNA破坏信号通路的图。如果在DSB部位发生新中心粒形成，则将代表一种以前未知的机制，可保护细胞免受基因组不稳定性，以应对DNA损伤。