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: 10605363
• 负责人: KATSUMI KITAGAWA
• 金额: $ 23.25万
• 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 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10605363
• 关键词: Air Pollutants; Aneuploidy; BUB1 gene; Cell Cycle Progression; Cells; Centromere; Chemicals; Chromatin; Chromosome Segregation; Chromosome abnormality; Chromosomes; Compensation; Complex; DNA Damage; DNA Double Strand Break; DNA Repair; DNA damage checkpoint; DNA lesion; Deposition; Dimensions; Double Strand Break Repair; Environment; Event; Exposure to; Failure; Goals; Heavy Metals; Histone H3; Human; Immunofluorescence Immunologic; Kinetochores; Lesion; Malignant Neoplasms; Mammalian Cell; Mediating; Mediator; Microscopic; Microtubules; Mitosis; Mitotic; Modeling; Molecular; Monitor; Mus; Nature; Play; Proteins; Radiation; Regulation; Role; Shapes; Signal Transduction; Site; Stress; Structure; Testing; Variant; 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.

细胞暴露于环境因子，如辐射、重金属、空气污染物和诱变剂 化学物质，产生DNA双链断裂（DSB）和其他染色体损伤。等 环境诱导的染色体损伤是耐受的并最终通过复合物消除， 这种保守机制称为DNA损伤反应（DDR）。我们的长期目标是阐明 动排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 定义着丝粒蛋白A（CENP-A），一种组蛋白H3变体，作为这种串扰的关键介质的作用。 CENP-A是着丝粒特异性染色质的一种成分，对于着丝粒的组装至关重要， 连接染色体和纺锤体微管的蛋白质结构。CENP- A在着丝粒身份和动粒组装中起着至关重要的作用。重要的是，我们和其他人 令人惊讶的发现是CENP-A也定位于正常和永生化的人和小鼠的DNA DSB 细胞现有证据表明CENP-A在DSB修复中起作用，但其作用机制尚不清楚。 完成这一壮举仍有待确定。我们假设CENP-A使一种 DSB位点的假/动粒激活纺锤体检查点并延迟细胞周期进程， 损坏修复失败。我们提出以下具体目标来检验我们的假设：目标1：确定 CENP-A、BUB 1和其他蛋白质在DSB形成的复合物的结构和功能。我们 工作假设是，含有CENP-A的复合物形成一个“假动粒”， DSB，从而激活纺锤体检查点（监测着丝粒-微管附着） 当DDR不能像其它着丝粒蛋白那样及时消除DNA损伤时（CENP-N， CENP-T和CENP-U）和BUB 1（纺锤体检查点组分）被募集到DSB。我们将 系统地检查已知的动粒蛋白是否位于DSB位点， 免疫荧光（IF）显微镜分析。目的2：评估纺锤体检查点在 延迟DSB修复中的细胞周期进展。我们假设DSB诱导的假/动粒可以 激活纺锤体检查点，导致有丝分裂延迟，允许DNA修复。我们将首先确定 纺锤体检查点是否通过IF定位在DBS位点。我们将确定是否有丝分裂延迟 当DNA损伤检查点活性被抑制时，DSB诱导的细胞凋亡依赖于纺锤体检查点成分。 无托叶目的3：检查新着丝粒是否在DNA修复失败后形成。偶尔， 含有CENP-A的位点可能成为完整的新着丝粒，这将拯救染色体片段 通过产生新的带有新着丝粒的染色体作为生存机制。我们将 当DNA修复或DNA损伤检查点被破坏时，在DSB诱导后筛选新着丝粒。 妥协，我们将检查是否新着丝粒形成增加在这些条件下。