Senataxin resolves RNA:DNA hybrids forming at DNA double-strand breaks to prevent translocations

Sarah Cohen,Vincent Rocher,Gaëlle Legube,Nadine Puget,Philippe Pasero,Yvan Canitrot,Thomas Clouaire,Marion Aguirrebengoa,Yea-Lih Lin

doi:10.1038/s41467-018-02894-w

Abstract

Ataxia with oculomotor apraxia 2 (AOA-2) and amyotrophic lateral sclerosis (ALS4) are neurological disorders caused by mutations in the gene encoding for senataxin (SETX), a putative RNA:DNA helicase involved in transcription and in the maintenance of genome integrity. Here, using ChIP followed by high throughput sequencing (ChIP-seq), we report that senataxin is recruited at DNA double-strand breaks (DSBs) when they occur in transcriptionally active loci. Genome-wide mapping unveiled that RNA:DNA hybrids accumulate on DSB-flanking chromatin but display a narrow, DSB-induced, depletion near DNA ends coinciding with senataxin binding. Although neither required for resection nor for timely repair of DSBs, senataxin was found to promote Rad51 recruitment, to minimize illegitimate rejoining of distant DNA ends and to sustain cell viability following DSB production in active genes. Our data suggest that senataxin functions at DSBs in order to limit translocations and ensure cell viability, providing new insights on AOA2/ALS4 neuropathies.

Highlights

Ataxia with oculomotor apraxia 2 (AOA-2) and amyotrophic lateral sclerosis (ALS4) are neurological disorders caused by mutations in the gene encoding for senataxin (SETX), a putative RNA:DNA helicase involved in transcription and in the maintenance of genome integrity
4 hydroxytamoxifen (4OHT) treatment induces the relocalisation of a stably expressed restriction enzyme (AsiSI) that in turn triggers the production of multiple double-strand breaks (DSBs) at annotated positions across the genome, in a homogeneous manner in the cell population allowing the use of chromatin immunoprecipitation (ChIP)[30]
We discovered that senataxin is recruited at DSBs induced in active loci, where it removes RNA:DNA hybrids forming in cis to broken loci

Summary

Introduction

Ataxia with oculomotor apraxia 2 (AOA-2) and amyotrophic lateral sclerosis (ALS4) are neurological disorders caused by mutations in the gene encoding for senataxin (SETX), a putative RNA:DNA helicase involved in transcription and in the maintenance of genome integrity. The exact mechanism that leads to such R-loops or/and RNA:DNA hybrids formation remains unclear and may either relate to the transcriptional extinction observed at damaged sites or to de novo RNA PolII loading at DNA ends and subsequent RNA production at the break point, two features that have been previously proposed to occur in many organisms In order to gain insights into R-loops biology at DSBs, here we set to assess a potential function of senataxin at sites of DNA DSBs. Using ChIP-seq and DRIP-seq, we uncovered that senataxin is recruited at DSBs induced in transcriptionally active genes, which exhibit RNA:DNA hybrids accumulation following DSB induction. It promotes Rad[51] foci formation, counteracts translocations and sustains viability following DSB production in active genes, identifying a crucial and unanticipated role for senataxin in DSB repair

Methods

Results

Conclusion