EXOSC10 is required for RPA assembly and controlled DNA end resection at DNA double-strand breaks

Judit Domingo-Prim,Martin Endara-Coll,Sonia Jimeno,Neus Visa,Rosario Prados-Carvajal,Pablo Huertas,Marc R Friedländer,Franziska Bonath

doi:10.1038/s41467-019-10153-9

Abstract

The exosome is a ribonucleolytic complex that plays important roles in RNA metabolism. Here we show that the exosome is necessary for the repair of DNA double-strand breaks (DSBs) in human cells and that RNA clearance is an essential step in homologous recombination. Transcription of DSB-flanking sequences results in the production of damage-induced long non-coding RNAs (dilncRNAs) that engage in DNA-RNA hybrid formation. Depletion of EXOSC10, an exosome catalytic subunit, leads to increased dilncRNA and DNA-RNA hybrid levels. Moreover, the targeting of the ssDNA-binding protein RPA to sites of DNA damage is impaired whereas DNA end resection is hyper-stimulated in EXOSC10-depleted cells. The DNA end resection deregulation is abolished by transcription inhibitors, and RNase H1 overexpression restores the RPA recruitment defect caused by EXOSC10 depletion, which suggests that RNA clearance of newly synthesized dilncRNAs is required for RPA recruitment, controlled DNA end resection and assembly of the homologous recombination machinery.

Highlights

The exosome is a ribonucleolytic complex that plays important roles in RNA metabolism
Our results identify RNA clearance at double-strand breaks (DSBs) as a step in the homologous recombination (HR) pathway that is required for the assembly of RPA onto the resected ssDNA, which in turn is a prerequisite for controlled DNA resection, RAD51 replacement and DNA repair by HR
The results reported above support a model in which EXOSC10 degrades dilncRNAs, and that dilncRNA degradation is required for RPA recruitment, the assembly of the HR machinery and to limit DNA end resection

Summary

Introduction

The exosome is a ribonucleolytic complex that plays important roles in RNA metabolism. We show that the exosome is necessary for the repair of DNA double-strand breaks (DSBs) in human cells and that RNA clearance is an essential step in homologous recombination. The MRE11-RAD50-NBS1 (MRN) complex recognizes the DSB and recruits the ATM/ATR kinases, which are responsible for the phosphorylation of the H2AX variant histone at Ser[137]. This phosphorylated histone, γH2AX, acts as a recruitment platform for adaptor proteins and promotes chromatin remodeling to increase the accessibility of the DDR effectors[2,3]. RPA is eventually replaced by the strand-exchange factor RAD51, a central player in HR that directs sister-chromatid strand invasion[7]

Methods

Results

Conclusion