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

细胞暴露于环境因素，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) 和其他染色体损伤。这样的 环境引起的染色体损伤可以被耐受并最终通过复杂的消除， 保守机制称为DNA损伤反应（DDR）。我们的长期目标是阐明 着丝粒介导的有丝分裂调节和 DDR 之间的分子串扰。我们特别努力 定义了着丝粒蛋白 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组成部分，对于着丝粒的组装至关重要， 提供染色体和纺锤体微管之间连接的蛋白质结构。 CENP- A 在着丝粒身份和着丝粒组装中起着至关重要的作用。重要的是，我们和其他人已经做出了 令人惊讶的发现是，CENP-A 也定位于正常和永生化人类和小鼠的 DNA DSB 细胞。现有证据表明 CENP-A 在 DSB 修复中发挥作用，但其机制 能否完成这一壮举仍有待确定。我们假设 CENP-A 成核形成 DSB 位点上的假/动粒可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、BUB1 和 DSB 上其他蛋白质形成的复合物的结构和功能。我们的 工作假设是，含有 CENP-A 的复合物形成一个“伪动粒”，在 DSB 随即激活纺锤体检查点（监控动粒-微管附着） 当DDR无法像其他着丝粒蛋白（CENP-N、 CENP-T 和 CENP-U）和 BUB1（纺锤体检查点成分）被招募到 DSB。我们会 系统地检查已知的动粒蛋白是否位于 DSB 位点 免疫荧光（IF）显微分析。目标 2：评估主轴检查点在 延迟 DSB 修复中的细胞周期进程。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，从而实现 DNA 修复。我们首先会确定 纺锤体检查点是否通过 IF 定位在 DBS 站点。我们将确定有丝分裂是否延迟 当DNA损伤检查点活性降低时，DSBs诱导的DNA损伤依赖于纺锤体检查点成分 缺席。目标 3：检查 DNA 修复失败时是否形成新着丝粒。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 通过生成具有新着丝粒的新染色体作为生存机制，从而消除无着丝粒的情况。我们会 当 DNA 修复或 DNA 损伤检查点处于 DSB 诱导后筛选新着丝粒 受损，我们将检查在这些条件下新着丝粒形成是否增加。

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.