Role for RIF1-interacting partner DDX1 in BLM recruitment to DNA double-strand breaks

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Human Rap1-interacting factor 1 (RIF1) is crucial for DNA double-strand break (DSB) repair, acting downstream of 53BP1.
• RIF1 has established roles in B-cell class switch recombination and inhibiting end resection in BRCA1-defective cells.

Purpose of the Study:

• To investigate the interaction and functional relationship between RIF1 and DEAD Box 1 (DDX1), an RNA helicase involved in DSB repair.
• To elucidate the distinct and shared roles of RIF1 and DDX1 in the homologous recombination pathway and DSB repair mechanisms.

Main Methods:

• Co-immunoprecipitation and co-localization studies to assess RIF1-DDX1 interaction and cellular localization.
• Depletion studies using RNA interference to evaluate the impact of RIF1 on DDX1 recruitment and function.
• Analysis of chromatin loading of Bloom syndrome helicase (BLM) and assessment of nucleic acid requirements for DDX1 and RIF1 accumulation at DSBs.

Main Results:

• DDX1 interacts with RIF1 and co-localizes with it throughout interphase.
• RIF1 depletion abolishes DDX1 recruitment to DSBs and its facilitation of homologous recombination.
• Both RIF1 and DDX1 are required for Bloom syndrome helicase (BLM) chromatin loading to DSBs, independently of 53BP1.
• DDX1 requires RNA-DNA hybrids, while RIF1 requires single-strand RNA for accumulation at DSBs.

Conclusions:

• DDX1 and RIF1 exhibit both convergent and divergent roles in DSB repair.
• The distinct nucleic acid requirements suggest complex regulatory mechanisms for these factors at DSB sites.
• These findings may explain why RIF1 depletion incompletely rescues homologous recombination defects in BRCA1-deficient cells.