Role of nuclear architecture in the spatial and temporal dynamics of heterochromatin repair

摘要 提高我们对着丝粒周围异染色质修复的认识是一项对改善 人类健康：异染色质是一个特征不佳的区域，占人类的近三分之一 基因组；该区域的双链断裂(DSB)修复失败不仅影响特定的基因，而且 全基因组范围的稳定性；在这里失败是一个高风险，因为丰富的重复序列 这是这个领域的特点。尽管描述这些过程具有基础性的重要性，DSB 异染色质的修复机制大多是未知的。我们最近发现了一条特殊的路径 促进异染色质中忠实的同源重组(HR)修复，同时防止大量 基因组不稳定。我们发现了Smc5/6复合体在这一途径中的关键作用，但这个复合体是如何 参与异染色质修复尚不清楚。解除对异染色质修复的管制可能是 最被低估和最强大的肿瘤发生来源，并确定涉及的成分是 对了解癌症病因和制定更有效的治疗策略至关重要 干预。为了深入了解Smc5/6在异染色质修复中的作用，我们使用质谱学方法 确定这一综合体的新的相互作用者，这将在本提案中进一步研究。我们的中央 假设修复发生在三个步骤：HR异常进展的初始阶段 在异染色质结构域内被抑制；修复位点重新定位到核的第二阶段 外周；第三阶段，特征是移除核内HR进展的阻滞物 外围设备。我们将结合丰富的成像、遗传和生化方法在果蝇细胞和 生物体识别这些步骤中涉及的分子靶标，并确定它们在空间和 异染色质修复的时间调节。这项研究的预期积极结果包括第一个 保护异染色质免受大规模基因组伤害的分子机制的系统鉴定 重新安排，使人力资源修复得以成功完成。这些研究也有望阐明 核结构和动力学、修复进程、RNAi沉默途径之间缺失的联系，以及 重复DNA序列的稳定性。这些结果将产生重要的积极影响， 正常细胞用来保护基因组免受环境威胁的关键保障机制。 这些途径的突变会导致基因组不稳定、肿瘤发生和寿命缩短。因此，我们 预计拟议的研究和未来的研究将在早期，在预防方面引发令人兴奋的进展 检测和治疗癌症和其他与基因组不稳定和衰老有关的人类疾病- 相关的障碍。

SUMMARY Advancing our knowledge of pericentromeric heterochromatin repair is a high impact investment for improving human health: heterochromatin is a poorly characterized region that comprises nearly a third of the human genome; double-strand break (DSB) repair failures in this region affect not just specific genes but also genome-wide stability; and failures here are a high risk because of the abundance of repeated sequences that characterizes this domain. In spite of the foundational importance of characterizing these processes, DSB repair mechanisms in heterochromatin are mostly unknown. We recently discovered a specialized pathway that promotes faithful homologous recombination (HR) repair in heterochromatin while preventing massive genome instability. We discovered a critical role of the Smc5/6 complex in this pathway, but how this complex participates in heterochromatin repair is unknown. Deregulation of heterochromatin repair is likely one of the most underestimated and powerful sources of tumorigenesis, and identifying the components involved is essential for understanding cancer etiology and developing more effective strategies for therapeutic intervention. To gain insight into the role of Smc5/6 in heterochromatin repair, we used mass spectroscopy to identify new interactors of this complex, which will be further investigated in this proposal. Our central hypothesis is that repair occurs in three steps: an initial phase when abnormal progression of HR is suppressed inside the heterochromatin domain; a second phase when repair sites relocalize to the nuclear periphery; and a third phase characterized by the removal of the block to HR progression at the nuclear periphery. We will combine a wealth of imaging, genetic and biochemical approaches in Drosophila cells and organisms to identify the molecular targets involved in these steps, and determine their role in the spatial and temporal regulation of heterochromatin repair. Expected positive outcomes of this research include the first systematic identification of the molecular machinery that protects heterochromatin from massive genome rearrangements, enabling successful completion of HR repair. These studies are also expected to illuminate missing links between nuclear architecture and dynamics, repair progression, RNAi silencing pathways, and the stability of repeated DNA sequences. These results will have an important positive impact by identifying crucial safeguard mechanisms used by normal cells to protect the genome from environmental threats. Mutations in these pathways result in genome instability, tumorigenesis, and reduced life span. Thus, we expect that the proposed studies and future research will trigger exciting advancements in the prevention, early detection, and treatment of cancer and other human diseases associated with genome instability and aging- related disorders.