RAP80 Phase Separation at DNA Double-Strand Break Promotes BRCA1 Recruitment and Tumor Radio-Resistance

Abstract

RAP80 has been characterized as a component of the BRCA1-A complex and is responsible for the recruitment of BRCA1 to DNA double-strand breaks (DSBs). However, we and others found that the recruitment of RAP80 and BRCA1 are not absolutely temporally synchronized, indicating that other mechanisms, apart from physical interaction, may be implicated. Recently, we and other groups have reported that liquid-liquid phase separation (LLPS) is a pivotal mechanism underlying DNA repair factors condensation at DSBs and their function. In this study, we aim to disclose whether RAP80 undergoes LLPS at DSBs and whether it is required for BRCA1 recruitment. To verify RAP80 is an LLPS protein and its function in DNA damage response (DDR): (1) candidate-mEGFP fusion protein formed condensates in cells and showed fluorescence recovery after photobleaching (FRAP); (2) candidate protein was expressed in Escherichia coli and purified with GST; (3) intrinsically disordered region (IDR) of RAP80 were predicted and tested in cell and in vitro; (4) lentivirus were used to construct RAP80-Knock out (KO) and RAP80 re-expression cell lines; (5) length gradient K63 poly-ubiquitin chains were chemically synthesized and incubated with RAP80 protein in vitro; (6) BRCA1 and RAP80 location were determined through immunofluorescence; (7) RAP80 protein expression in tissue was determined by IHC staining. Thin layer scanning and 3D reconstruction of the RAP80-mEGFP-expressing cells under a fluorescence microscope showed that RAP80-mEGFP formed spherical condensates with fast FRAP. Observation of purified proteins revealed that GST-RAP80-mEGFP protein formed liquid-like droplets, presenting as a FRAP and the fusion event among adjacent droplets. PEG-8000 and Ficol-400 strengthened the formation of GST-RAP80-mEGFP droplets in vitro. Later, we used a previously developed optoIDR tool to verify that IDR1 (1-254aa) is critical for RAP80 LLPS. To investigate whether the interaction between RAP80 and K63 poly-ubiquitin chains could enhance the condensation of RAP80, we chemically synthesized K63 ubiquitin chains and incubated them with purified GST-RAP80-mCherry proteins. The results showed that supplementation of ubiquitin multipolymer (poly-ubiquitin) significantly induced the LLPS of RAP80, and the ability of RAP80 condensates formation potency was positively correlated with the length of the ubiquitin chain. Consistent with their LLPS capacity, RAP80-WT-mEGFP, RAP80-(IDR1+AIR)-mEGFP groups showed prominent BRCA1 foci, while RAP80-IDR1-mEGFP and RAP80-(SIM+UIM)-mEGFP groups showed delayed BRCA1 recruitment. In rectal cancer tissues, positive staining of the RAP80 protein was mainly observed in the nucleus of cancer cells and high RAP80 expression was correlated with a shorter overall survival time. RAP80 undergoes LLPS to form liquid-like condensates at DSB sites, which is important for BRCA1 recruitment and enhances tumor radio-resistance.