Regulation of DNA repair by FET family protein phase separation

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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.**