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

细胞暴露于环境因素，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。是这样的 环境引起的染色体损伤是可以容忍的，并最终通过一种复合体， 保守的机制称为DNA损伤反应(DDR)。我们的长期目标是澄清 动粒介导的有丝分裂调控和DDR之间的分子串扰。特别是，我们努力做到 确定着丝粒蛋白A(CENP-A)的作用，它是组蛋白H3的变体，是这种串扰的关键媒介。 CENP-A是着丝粒特异染色质的一种成分，着丝粒特异性染色质是动粒组装所必需的， 连接染色体和纺锤体微管的蛋白质结构。CENP- A在着丝粒识别和着丝粒组装中起着至关重要的作用。重要的是，我们和其他人已经取得了 令人惊讶的发现，CENP-A也定位于正常和永生化的人和小鼠的DNADSB 细胞。现有证据表明CENP-A在DSB修复中起作用，但其机制 完成这一壮举还有待确定。我们假设CENP-A形成了一个 DSB位点的伪/动粒激活纺锤体检查点并延缓细胞周期进展 损坏修复失败。我们提出了以下具体目标来检验我们的假设：目标1：确定 CENP-A、Bub1和其他蛋白质在DSB形成的复合体的结构和功能。我们的 工作假说是，一个含有CENP-A的复合体形成一个“假着丝点”，它在 DSB，于是它激活纺锤体检查点(该检查点监控着动粒-微管连接) 当DDR不能像其他着丝粒蛋白(CENP-N， CENP-T、CENP-U)和纺锤体检查点组件Bub1被招募到DSB。我们会 系统研究已知的动粒蛋白是否定位于DSB位点 免疫荧光(IF)显微镜分析。目标2：评估磁盘轴检查点在 在DSB修复中延缓细胞周期进程。我们假设DSB诱导的假/动律可以 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 是否通过IF在DBS站点本地化磁盘轴检查点。我们将确定有丝分裂延迟是否 DSB诱导的DNA损伤检查点活动依赖于纺锤体检查点组件 缺席。目的3：检测DNA修复失败后是否形成新着丝粒。偶尔， 含有CENP-A的基因座可能会变成完整的新着丝粒，这将挽救染色体片段 没有着丝粒，通过产生新的染色体和新的着丝粒作为生存机制。我们会 当DNA修复或DNA损伤检查点为 我们将检查在这些条件下新着丝粒的形成是否增加。

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.