Department of Molecular and Computational Biology, University of Southern California, Los Angeles 90089, USA.
Exp Biol Med (Maywood). 2019 Nov;244(15):1362-1371. doi: 10.1177/1535370219862282. Epub 2019 Jul 31.
Repairing DNA double-strand breaks is particularly challenging in pericentromeric heterochromatin, where the abundance of repeated sequences exacerbates the risk of ectopic recombination. In Kc cells, accurate homologous recombination repair of heterochromatic double-strand breaks relies on the relocalization of repair sites to the nuclear periphery before Rad51 recruitment and strand invasion. This movement is driven by Arp2/3-dependent nuclear actin filaments and myosins’ ability to walk along them. Conserved mechanisms enable the relocalization of heterochromatic repair sites in mouse cells, and defects in these pathways lead to massive ectopic recombination in heterochromatin and chromosome rearrangements. In polytene chromosomes, extensive DNA movement is blocked by a stiff structure of chromosome bundles. Repair pathways in this context are poorly characterized, and whether heterochromatic double-strand breaks relocalize in these cells is unknown. Here, we show that damage in heterochromatin results in relaxation of the heterochromatic chromocenter, consistent with a dynamic response. Arp2/3, the Arp2/3 activator Scar, and the myosin activator Unc45, are required for heterochromatin stability in polytene cells, suggesting that relocalization enables heterochromatin repair also in this tissue. Together, these studies reveal critical roles for actin polymerization and myosin motors in heterochromatin repair and genome stability across different organisms and tissue types.
Heterochromatin relies on dedicated pathways for ‘safe’ recombinational repair. In mouse and fly cultured cells, DNA double-strand break repair requires the movement of damaged sites away from the heterochromatin ‘domain’ nuclear actin filaments and myosins. Here, we explore the importance of these pathways in salivary gland cells, which feature a stiff bundle of endoreduplicated polytene chromosomes. Repair pathways in polytene chromosomes are largely obscure and how nuclear dynamics operate in this context is unknown. We show that heterochromatin relaxes in response to damage, and relocalization pathways are necessary to prevent abnormal repair and promote the stability of heterochromatic sequences. These results deepen our understanding of DNA damage response mechanisms in polytene chromosomes, revealing unexpected dynamics. It also provides a first understanding of nuclear dynamics responding to replication damage and rDNA breaks, providing a new understanding of the importance of nuclear architecture in genome stability. We expect these discoveries will shed light on tumorigenic processes, including therapy-induced cancer relapses.
在着丝粒异染色质中，修复 DNA 双链断裂尤其具有挑战性，大量重复序列的存在加剧了异位重组的风险。在 Kc 细胞中，异染色质双链断裂的同源重组修复依赖于 Rad51 募集和链入侵前修复位点向核周的重新定位。这种运动是由依赖于 Arp2/3 的核肌动蛋白丝和肌球蛋白沿肌动蛋白丝行走的能力驱动的。保守的机制使小鼠细胞中异染色质修复位点的重新定位成为可能，而这些途径的缺陷会导致异染色质和染色体重排中的大量异位重组。在多线染色体中，广泛的 DNA 运动被染色体束的刚性结构所阻断。在这种情况下，修复途径的特征描述很差，并且这些细胞中的异染色质双链断裂是否重新定位尚不清楚。在这里，我们表明，异染色质中的损伤导致异染色质染色质中心的松弛，与动态反应一致。Arp2/3、Arp2/3 激活因子 Scar 和肌球蛋白激活因子 Unc45，是多线细胞中异染色质稳定性所必需的，这表明重新定位也使异染色质修复成为可能。总之，这些研究揭示了肌动蛋白聚合和肌球蛋白马达在不同生物体和组织类型的异染色质修复和基因组稳定性中的关键作用。
异染色质依赖于专门的途径进行“安全”的重组修复。在小鼠和苍蝇培养细胞中，DNA 双链断裂修复需要将受损部位从异染色质“域”核肌动蛋白丝和肌球蛋白移动开来。在这里，我们探索了这些途径在唾液腺细胞中的重要性，唾液腺细胞具有僵硬的内复制多线染色体束。多线染色体中的修复途径在很大程度上是不为人知的，核动力学在这种情况下是如何运作的也不清楚。我们表明，异染色质在受到损伤时会松弛，重新定位途径是防止异常修复和促进异染色质序列稳定所必需的。这些结果加深了我们对多线染色体中 DNA 损伤反应机制的理解，揭示了意想不到的动态。它还提供了对复制损伤和 rDNA 断裂反应的核动力学的初步理解，为核结构在基因组稳定性中的重要性提供了新的认识。我们预计这些发现将为肿瘤发生过程提供启示，包括治疗诱导的癌症复发。
