An OB-fold complex controls the repair pathways for DNA double-strand breaks

Area of Science:

• Molecular Biology
• DNA Repair Mechanisms
• Cellular Signaling

Background:

• 53BP1 antagonizes BRCA1-mediated homologous recombination (HR) and promotes non-homologous end joining (NHEJ) via its downstream proteins RIF1, PTIP, and REV7.
• The precise mechanism by which 53BP1 and its associated factors regulate DNA double-strand break (DSB) repair remains incompletely understood.

Purpose of the Study:

• To elucidate the role of REV7-associated proteins in DNA DSB repair pathways.
• To identify novel components involved in the interplay between HR and NHEJ.
• To characterize the function of FAM35A and C20ORF196 in DNA repair.

Main Methods:

• Co-immunoprecipitation to identify protein complexes.
• Electrophoretic mobility shift assays (EMSAs) for DNA binding.
• In vivo recruitment assays to DNA double-strand breaks (DSBs).
• Epistasis analysis to determine pathway relationships.
• Analysis of DNA repair defects in BRCA1-mutant cells.

Main Results:

• REV7 forms a stable complex with FAM35A and C20ORF196.
• FAM35A exhibits preferential binding to single-stranded DNA (ssDNA) in vitro.
• The REV7-FAM35A-C20ORF196 complex is recruited to DSBs downstream of RIF1 in vivo.
• FAM35A and C20ORF196 function in the same NHEJ pathway as RIF1.
• Inactivation of FAM35A or C20ORF196 suppresses HR defects and reduces DNA end resection in BRCA1-mutant cells, indicating they prevent end resection.

Conclusions:

• A novel REV7-FAM35A-C20ORF196 complex is identified, playing a critical role in DNA DSB repair.
• This complex binds and protects broken DNA ends, thereby promoting the NHEJ pathway.
• FAM35A and C20ORF196 act as suppressors of DNA end resection, influencing the balance between NHEJ and HR repair.