The role of CENP-A in the response to DNA double-strand breaks

CENP-A 在 DNA 双链断裂反应中的作用

• 批准号: 10443414
• 负责人: KATSUMI KITAGAWA
• 金额: $ 19.38万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-07 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10443414
• 关键词: Air Pollutants; Aneuploidy; BUB1 gene; Cell Cycle Progression; Cells; Centromere; Chemicals; Chromatin; Chromosome Segregation; Chromosome abnormality; Chromosomes; Complex; DNA Damage; DNA Double Strand Break; DNA Repair; DNA damage checkpoint; DNA lesion; Deposition; Dimensions; Double Strand Break Repair; Event; Exposure to; Failure; Goals; Heavy Metals; Histone H3; Human; Immunofluorescence Immunologic; Kinetochores; Lesion; Malignant Neoplasms; Mammalian Cell; Mediating; Mediator of activation protein; Microscopic; Microtubules; Mitosis; Mitotic; Modeling; Molecular; Monitor; Mus; Nature; Play; Proteins; Radiation; Regulation; Role; Signal Transduction; Site; Stress; Structure; Testing; Time; Variant; base; centromere protein A; environmental agent; environmental mutagens; experimental study; high risk; novel; overexpression; preservation; recruit; response

Exposure of cells to environmental agents, such as radiation, heavy metals, air pollutants and mutagenic chemicals, generates DNA double-strand breaks (DSBs) and other chromosomal lesions. Such environmentally induced chromosomal lesions are tolerated and ultimately eliminated via a complex, conserved mechanism termed the DNA damage response (DDR). Our long-term goal is to elucidate the molecular crosstalk between kinetochore-mediated mitotic regulation and the DDR. In particular, we strive to define the role of centromere protein A (CENP-A), a histone H3 variant, as a key mediator of this crosstalk. CENP-A is a constituent of the centromere-specific chromatin essential for the assembly of the kinetochore, a proteinaceous structure that provides the connection between chromosomes and spindle microtubules. CENP- A plays a crucial role in centromere identity and kinetochore assembly. Importantly, we and others have made the surprising finding that CENP-A also localizes to DNA DSBs in normal and immortalized human and mouse cells. The available evidence suggests that CENP-A functions in DSB repair, but the mechanism by which it accomplishes this feat remains to be determined. We hypothesize that CENP-A nucleates the formation of a pseudo/kinetochore at DSB sites to activate the spindle checkpoint and delay cell cycle progression when DNA damage repair fails. We propose the following Specific Aims to test our hypothesis: Aim 1: Determine the structure and function of the complex formed by CENP-A, BUB1, and other proteins at DSBs. Our working hypothesis is that a CENP-A-containing complex forms a “pseudo kinetochore” that assembles at DSBs whereupon it activates the spindle checkpoint (which monitors kinetochore-microtubule attachment) when DDR fails to eliminate the DNA lesions in a timely fashion as other centromere proteins (CENP-N, CENP-T, and CENP-U) and BUB1, a spindle checkpoint component, are recruited to DSBs. We will systematically examine whether known kinetochore proteins are localized at the DSB sites by immunofluorescence (IF) microscopic analysis. Aim 2: Assess the role of the spindle checkpoint in delaying cell cycle progression in DSB repair. We hypothesize that DSB-induced pseudo/kinetochores can activate the spindle checkpoint to cause a delay in mitosis, allowing DNA repair. We will first determine whether spindle checkpoints are localized at DBS sites by IF. We will determine whether the mitotic delay induced by DSBs is reliant on spindle checkpoint components when the DNA damage checkpoint activities are absent. Aim 3: Examine whether neocentromeres are formed upon failure of DNA repair. Occasionally, CENP-A-containing loci may become intact neocentromeres, which would rescue chromosome fragments without centromeres by generating new chromosomes with neocentromeres as a survival mechanism. We will screen for neocentromeres after DSB induction when DNA repair or the DNA damage checkpoint is compromised, and we will examine whether neocentromere formation increases under these conditions.

将细胞暴露于环境药物，例如辐射，重金属，空气污染物和诱变剂 化学物质会产生DNA双链断裂（DSB）和其他染色体病变。这样的 环境诱导的染色体病变被耐受，并最终通过复合物消除 保守机制称为DNA损伤反应（DDR）。我们的长期目标是阐明 动力学介导的有丝分裂调节和DDR之间的分子串扰。特别是，我们努力 将Centromere蛋白A（CENP-A）（一种组蛋白H3变体）定义为该串扰的关键介体的作用。 CENP-A是Centromere特异性染色质的构成，对于组装动力学所必需 蛋白质结构，提供染色体和纺锤微管之间的连接。 CENP- A在Centromere身份和动力学组装中起着至关重要的作用。重要的是，我们和其他人做了 令人惊讶的发现，CENP-A在正常和永生的人和小鼠中也定位于DNA DSB 细胞。现有证据表明CENP-A在DSB维修中起作用，但其机制 完成这一壮举还有待确定。我们假设CENP-A核形成了 DSB位点的伪/动力学，以激活纺锤体检查点并延迟细胞周期进程 损坏维修失败。我们提出以下特定目的来检验我们的假设：目标1：确定 CENP-A，BUB1和其他蛋白质在DSB中形成的复合物的结构和功能。我们的 工作假设是，含CENP-A的复合物形成了一个“伪动物学”，该复合物组装在 dsbs随后激活主轴检查点（该检查点可监视kinetochore-microubule附件） 当DDR无法及时消除DNA病变，因为 CENP-T，CENP-U）和BUB1（纺锤体检查点组件）被招募到DSB。我们将 系统地检查已知的动力学蛋白是否位于DSB位置 免疫荧光（IF）显微镜分析。目标2：评估主轴检查站的作用 延迟DSB修复中的细胞周期进程。我们假设DSB诱导的伪/动物学可以 激活主轴检查点，导致有丝分裂的延迟，从而允许DNA修复。我们将首先确定 主轴检查点是否位于DBS网站上。我们将确定是否有丝分裂延迟 当DNA损伤检查点活动为 缺席的。 AIM 3：检查在DNA修复失败后是否形成新中心粒。偶尔， 含Cenp-a的基因座可能成为完整的新中心粒子，它将挽救染色体片段 通过将新的染色体作为一种生存机制产生新的染色体，没有中心粒。我们将 DSB诱导后的新中心粒筛选DNA修复或DNA损伤检查点为 受到妥协，我们将检查在这些条件下的新中心粒粒子形成是否增加。