Regulation of DNA repair by FET family protein phase separation

FET 家族蛋白相分离调节 DNA 修复

• 批准号: RGPIN-2019-07088
• 负责人: Lee, Hyun
• 金额: $ 2.7万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=744135
• 关键词: Regulation; DNA; repair; FET; family

Cellular reactions often occur in dedicated compartments or organelles that selectively concentrate or exclude certain molecules. A large class of organelles, like nucleoli and RNA processing bodies, segregate their contents without lipid membrane barriers. Instead, they form through a process known as liquid-liquid phase separation driven by transient inter-molecular interactions, like oil droplets in water. From extensive in vitro and structural studies, we are now beginning to understand the molecular signatures that drive the assembly and the properties of phase-separated compartments, which are also called biomolecular condensates. Yet, several key questions remain unanswered: what is the functional role and significance of condensate assembly on cellular processes, and how are their assembly and disassembly regulated to support this function? Recent studies from myself and others have shown that the FET family of proteins (FUS, EWS, TAF15) arrives at early stages of DNA damage sites and form liquid-like condensates. Their function has been implicated in efficient repair of double-strand DNA breaks, but how they facilitate this process is not known. Using quantitative imaging techniques and cellular and protein tools I have established, I aim to identify the factors that regulate the assembly and disassembly of FET condensates at DNA damage sites and determine the role of the condensates in double-strand DNA break repair. Using FUS first as a model, we will test the following hypothesis in three aims: 1) FET condensate formation at DNA damage sites is regulated by post-translational modifications and by the concentration of certain nucleotide species (Aim 1); 2) FET condensate dissolution and exclusion at DNA damage sites is regulated by the subsequent arrival of double-strand break proteins such as ATM and TP53BP1 (Aim 2); 3) FET condensates enhance DNA repair by amplifying the concentration of certain repair proteins, while excluding others (Aim 3). In the longer term, we will test the hypothesis that DNA damage sites orchestrate sequential formation and disassembly of a series of distinct liquid condensates that promote specific steps of DNA repair. Our findings will reveal novel mechanisms regulating DNA repair and provide valuable insights into how biomolecular phase separation promotes cell fitness.

细胞反应通常发生在选择性浓缩或排除某些分子的专用隔室或细胞器中。一大类细胞器，如核仁和RNA加工体，在没有脂质膜屏障的情况下分离其内容物。相反，它们是通过一个被称为液-液相分离的过程形成的，该过程由瞬时分子间相互作用驱动，就像水中的油滴一样。通过广泛的体外和结构研究，我们现在开始了解驱动组装的分子特征和相分离隔室的性质，这些隔室也称为生物分子凝聚物。然而，有几个关键问题仍然没有答案：什么是功能的作用和意义上的冷凝物组装的细胞过程，以及如何调节它们的组装和拆卸，以支持这一功能？我和其他人最近的研究表明，FET蛋白家族（FUS，EWS，TAF 15）到达DNA损伤位点的早期阶段，并形成液体状冷凝物。它们的功能与双链DNA断裂的有效修复有关，但它们如何促进这一过程尚不清楚。使用定量成像技术和细胞和蛋白质的工具，我已经建立，我的目标是确定调节FET冷凝物在DNA损伤位点的组装和拆卸的因素，并确定冷凝物在双链DNA断裂修复中的作用。首先以FUS为模型，我们将从三个方面验证以下假设：1）DNA损伤位点的FET凝聚物的形成受翻译后修饰和某些核苷酸种类的浓度的调节（Aim 1）; 2）DNA损伤位点的FET凝聚物的溶解和排斥受随后到达的双链断裂蛋白如ATM和TP 53 BP 1的调节（Aim 2）; 3）FET缩合物通过放大某些修复蛋白的浓度而排除其他蛋白来增强DNA修复（目的3）。从长远来看，我们将测试DNA损伤位点协调一系列不同液体冷凝物的顺序形成和分解的假设，这些冷凝物促进DNA修复的特定步骤。我们的研究结果将揭示调节DNA修复的新机制，并为生物分子相分离如何促进细胞适应性提供有价值的见解。