FUS-dependent liquid-liquid phase separation is important for DNA repair initiation

Area of Science:

• Cellular biology
• Molecular genetics
• Biochemistry

Background:

• RNA-binding proteins (RBPs) play key roles in the DNA damage response (DDR).
• The RBP FUS is involved in RNA metabolism and DNA repair.
• FUS undergoes reversible liquid-liquid phase separation (LLPS) in vitro.

Purpose of the Study:

• To investigate the role of FUS-dependent LLPS in the initiation of the DDR.
• To determine if FUS-LLPS is necessary for recruiting DDR factors to DNA damage sites.

Main Methods:

• Laser microirradiation in FUS-knockout cells.
• Assessment of DDR factor recruitment (KU80, NBS1, 53BP1, SFPQ).
• Use of LLPS inhibitors and LLPS-deficient FUS variants.
• Analysis of γH2AX foci formation and arrangement using superresolution microscopy.

Main Results:

• FUS is required for the recruitment of DDR factors (KU80, NBS1, 53BP1, SFPQ) to DNA damage sites.
• Impaired recruitment and γH2AX foci formation were observed with LLPS inhibitors or LLPS-deficient FUS.
• Absence of FUS disrupts the higher-order clustering of γH2AX nanofoci.

Conclusions:

• FUS-dependent LLPS is essential for the early activation of the DDR.
• LLPS is critical for the proper assembly of double-strand break (DSB) repair complexes.
• FUS-mediated LLPS is a key mechanism in orchestrating the cellular response to DNA damage.