The role of RNA in the liquid-liquid phase separation of 53BP1 and genome stability

The synthesis of DNA damage-induced RNA at sites of DNA double-strand breaks (DSBs) assists the DNA damage response (DDR) by promoting the focal recruitment of its factors at DSBs. 53BP1, a crucial component of the non-homologous end joining (NHEJ) DNA repair pathway, is recruited to DSB repair foci in a transcription-dependent manner and exhibits liquid-like properties. However, the mechanisms governing the liquid-liquid phase separation (LLPS) of 53BP1 and its regulation by RNA remain unclear. To address this, we investigate in vitro the molecular determinants of 53BP1 that drive its LLPS. This thesis highlights the importance of RNA in driving the condensation of 53BP1 by demonstrating the process in vitro using a recombinant fragment of 53BP1 that contains its oligomerizaIon domain and RNA-binding RGG motif. This fragment, termed F6, is shown to undergo LLPS with RNA in vitro and display liquid-like properties such as growth, fusion, and dissolution upon exposure to chaotropic agents like ammonium acetate and 1,6-hexanediol. Additionally, F6 + RNA condensates rapidly dissolve upon the addition of RNaseA, and reform upon its inhibition followed by the re-addition of RNA. The F6 fragment also directly binds RNA through its RGG motif, losing its ability to do so and consequently form condensates upon its mutation. Lastly, it is shown that long RNA (>200 nt) drives the LLPS of F6 to a higher extent in comparison to short RNA (<200 nt). We therefore propose RNA to have an indispensable role in driving the LLPS of 53BP1, and consequently stimulating the DDR into proper functioning.