Regulation of DNA repair by FET family protein phase separation

细胞反应通常发生在有选择性地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，在没有脂膜屏障的情况下将其内容物分离。相反，它们是通过一种被称为液-液相分离的过程形成的，这种过程由瞬时的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，这也被称为生物分子凝聚物。然而，几个关键问题仍然没有得到回答：凝析油组装在细胞过程中的功能作用和意义是什么，它们的组装和分解是如何被调节以支持这一功能的？我和其他人最近的研究表明，FET家族蛋白质(FUS、EWS、TAF15)到达DNA损伤部位的早期阶段，并形成液状凝聚体。它们的功能与有效修复双链DNA断裂有关，但它们如何促进这一过程尚不清楚。利用定量成像技术以及我建立的细胞和蛋白质工具，我的目标是确定调控DNA损伤部位FET凝聚体组装和拆解的因素，并确定凝聚体在双链DNA断裂修复中的作用。以FUS First为模型，我们将在三个目标中检验以下假设：1)FET凝聚体在DNA损伤部位的形成受翻译后修饰和某些核苷酸物种的浓度的调节(目标1)；2)FET凝聚体在DNA损伤部位的溶解和排除受随后到达的双链断裂蛋白如ATM和TP53BP1的调节(目标2)；3)FET凝聚体通过放大某些修复蛋白的浓度而增强DNA修复，而排除其他修复蛋白(目标3)。从长远来看，我们将测试这一假设，即DNA损伤部位策划了一系列不同液体冷凝物的顺序形成和拆解，这些冷凝物促进了DNA修复的特定步骤。我们的发现将揭示调控DNA修复的新机制，并为生物分子相分离如何促进细胞健康提供有价值的见解。

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.