Regulation of DNA repair by FET family protein phase separation

细胞反应通常发生在专门的室室或细胞器中，它们选择性地浓缩或排除某些分子。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它们的内容物。相反，它们是通过一种称为液-液相分离的过程形成的，这种过程是由瞬时分子间相互作用驱动的，就像水中的油滴一样。从广泛的体外和结构研究中，我们现在开始了解驱动相分离隔室的组装和特性的分子特征，也称为生物分子凝聚物。然而，仍有几个关键问题没有得到解答：凝析油组装在细胞过程中的功能作用和意义是什么？它们的组装和拆卸是如何调节以支持这一功能的？******我和其他人最近的研究表明，FET家族的蛋白质（FUS， EWS, TAF15）到达DNA损伤位点的早期阶段并形成液体状凝聚体。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。利用我已经建立的定量成像技术和细胞和蛋白质工具，我的目标是确定在DNA损伤位点调节FET凝聚体的组装和拆卸的因素，并确定凝聚体在双链DNA断裂修复中的作用。首先使用FUS作为模型，我们将在三个目标中验证以下假设：1)DNA损伤位点的FET凝聚物形成受翻译后修饰和某些核苷酸种类的浓度调节（目的1）；2) FET凝聚物在DNA损伤位点的溶解和排除受随后到达的双链断裂蛋白如ATM和TP53BP1的调控（Aim 2）；3)场效应晶体管凝聚体通过放大某些修复蛋白的浓度来增强DNA修复，而排除其他修复蛋白（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凝聚物的顺序形成和分解，从而促进DNA修复的特定步骤的假设。我们的发现将揭示调节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

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.**