USP7 deubiquitinase promotes ubiquitin-dependent DNA damage signaling by stabilizing RNF168

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Histone ubiquitination by RNF168 is vital for DNA damage response (DDR).
• RNF168 recruits essential DDR factors like BRCA1 and 53BP1.
• The regulation of RNF168 stability is critical for efficient DNA repair signaling.

Purpose of the Study:

• To investigate the role of USP7 deubiquitinase in regulating RNF168 stability.
• To elucidate how USP7 affects RNF168-mediated ubiquitination and downstream DDR factor recruitment.
• To understand the mechanism by which USP7 controls RNF168 levels during DNA damage response.

Main Methods:

• Utilized cell-based assays to assess the impact of USP7 disruption on RNF168 stability and histone ubiquitination.
• Employed siRNA-mediated depletion and overexpression of USP7 and its mutants.
• Performed co-immunoprecipitation assays to confirm in vitro and in vivo binding of USP7 and RNF168.
• Assessed the formation of DNA damage-induced foci (e.g., UVRIF, IRIF) for key DDR proteins.

Main Results:

• USP7 disruption impairs H2A and UVR-induced γH2AX monoubiquitination and decreases RNF168 and BRCA1 levels.
• USP7 physically binds RNF168, and wild-type USP7 prevents UVR-induced RNF168 degradation.
• USP7 disruption compromises the formation of uH2A, polyubiquitinated H2AX/A, BRCA1, and 53BP1 foci.
• Ectopic expression of RNF168 rescues DDR foci formation in USP7-disrupted cells.

Conclusions:

• USP7 deubiquitinase plays a crucial role in stabilizing RNF168.
• USP7 regulates ubiquitin-dependent signaling pathways by controlling RNF168 stability.
• These findings reveal a novel regulatory mechanism in DNA damage response signaling mediated by USP7 and RNF168.