Ubiquitin-induced RNF168 condensation promotes DNA double-strand break repair

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• DNA double-strand breaks (DSBs) require rapid repair factor accumulation.
• Liquid-liquid phase separation (LLPS) is increasingly recognized as a mechanism facilitating DSB repair.
• RNF168, an E3 ubiquitin ligase, plays a role in DSB repair by ubiquitylating H2A.X.

Purpose of the Study:

• To investigate whether RNF168 undergoes LLPS at DSB sites.
• To elucidate the role of RNF168 LLPS in DNA repair dynamics and efficiency.

Main Methods:

• Cellular irradiation and imaging to observe RNF168 condensation.
• In vitro protein condensation assays with purified RNF168.
• Identification of the intrinsically disordered region responsible for condensation.
• Ubiquitination assays to assess RNF168 catalytic activity.
• Recruitment assays for DNA repair factors 53BP1 and BRCA1.

Main Results:

• RNF168 forms liquid-like condensates in the nucleus upon irradiation and in vitro.
• An intrinsically disordered region (amino acids 460-550) is critical for RNF168 condensation.
• K63-linked polyubiquitin chains significantly enhance RNF168 LLPS.
• RNF168 LLPS boosts H2A.X ubiquitination, creating a positive feedback loop.
• LLPS deficiency in RNF168 impairs 53BP1 and BRCA1 recruitment and delays DSB repair.

Conclusions:

• RNF168 undergoes K63-linked polyubiquitin-triggered LLPS at DSB sites.
• LLPS enhances RNF168's catalytic activity and recruitment of repair factors.
• RNF168-mediated LLPS is crucial for efficient DNA double-strand break repair.