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Formation of Neocentromere at a DSB Site

DSB 位点新着丝粒的形成

基本信息

批准号：
9244010
负责人：
KATSUMI KITAGAWA
金额：
$ 19.9万
依托单位：
UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-03-15 至 2019-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9244010
关键词：
Adverse effects Apoptotic Binding Cell Death Cell Survival Cells Centromere Chemical Agents Chromatin Chromatin Structure 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 base cancer cell cancer therapy centromere protein A chemotherapeutic agent experimental study genetic information inhibitor/antagonist insight neoplastic cell novel protein complex public health relevance radiosensitive repaired response segregation tumorigenesis

项目摘要

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（由申请人提供）：组蛋白 H3 变体、着丝粒蛋白 A (CENP-A) 也定位于 DNA 双链断裂 (DSB)，这意味着参与对 DNA 损伤的反应。 CENP-A 通常是着丝粒特异性染色质的组成部分，形成着丝粒组装的基础，着丝粒是一种蛋白质结构，包含许多保守的蛋白质复合物，充当染色体和纺锤体微管之间的界面。 CENP-A 在着丝粒身份和着丝粒组装中起着至关重要的作用，并且对于着丝粒和着丝粒功能至关重要，但为什么它应该被招募到 DSB 焦点尚不清楚。我们的工作假设是，CENP-A 被招募到 DSB，以创建 DNA 损伤特异性形式的染色质，DNA 损伤检查点/修复蛋白被招募到其中。基于这个想法，我们提出了一个新模型来描述未修复的 DSB 的重要生存机制。在这个模型中，未修复的 DNA 损伤会产生可能含有重要遗传信息的无着丝粒染色体片段。为了稳定地继承这些无着丝粒片段，必须在断裂的染色体片段上由CENP-A蛋白标记的DSB位点处形成新着丝粒。新着丝粒有利于微管附着和有丝分裂过程中的正确分离。我们假设 CENP-A 在 DSB 位点的掺入可导致新着丝粒的形成，以及化疗药物或放射治疗诱导的 DSB 修复失败后细胞的存活。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 在 DSB 站点定位的重要性。我们将把 DSB 引入缺乏 CENP-A 的细胞的染色体中，然后分析 DSB 位点的 DNA 损伤检查点活性和 DNA 修复动力学，并确定已知的 DNA 损伤检查点/修复蛋白向 DSB 位点的募集是否被取消。此外，我们将测量 DNA 修复活性以及 CENP-A 耗尽的细胞的存活率。目标 2. 确定 DSB 部位是否发生新着丝粒形成。我们假设新着丝粒或“伪”新着丝粒以 CENP-A 依赖性方式在 DSB 位点组装。 DSB 已鉴定出一些纺锤体组装检查点组件，但尚不清楚是否组装了可捕获微管的功能性动粒。为了检验这一假设，我们将进行免疫荧光显微镜检测所有着丝粒成分。我们还将检查当 DDR 通路失败时新着丝粒形成是否会增加。这些研究将为 CENP-A 在细胞存活中的作用提供新的见解。该研究项目将建立CENP-A在DDR中的新作用，并重塑DNA损伤信号通路的图谱。如果新着丝粒形成发生在 DSB 位点，它将代表一种以前未知的机制，用于保护细胞免受 DNA 损伤引起的基因组不稳定的影响。

项目成果

期刊论文数量（6）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A).

DOI：
10.3791/61138
发表时间：
2020-06
期刊：
Journal of visualized experiments : JoVE
影响因子：
0
作者：
Y. Niikura;Lei Fang;R. Kitagawa;Peizhao Li;Y. Xi;Ju You;Yan Guo;K. Kitagawa
通讯作者：
Y. Niikura;Lei Fang;R. Kitagawa;Peizhao Li;Y. Xi;Ju You;Yan Guo;K. Kitagawa

SGT1-HSP90 complex is required for CENP-A deposition at centromeres.