BRCA1 Recruitment to Transcriptional Pause Sites Is Required for R-Loop-Driven DNA Damage Repair

Elodie Hatchi,Konstantina Skourti-Stathaki,Steffen Ventz,Luca Pinello,Angela Yen,Kinga Kamieniarz-Gdula,Stoil Dimitrov,Shailja Pathania,Kristine M Mckinney,Matthew L Eaton,Manolis Kellis,Sarah J Hill,Giovanni Parmigiani,Nicholas J Proudfoot,David M Livingston

doi:10.1016/j.molcel.2015.01.011

Elodie Hatchi, Konstantina Skourti-Stathaki + Show 13 more

Open Access

https://doi.org/10.1016/j.molcel.2015.01.011

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Abstract

SummaryThe mechanisms contributing to transcription-associated genomic instability are both complex and incompletely understood. Although R-loops are normal transcriptional intermediates, they are also associated with genomic instability. Here, we show that BRCA1 is recruited to R-loops that form normally over a subset of transcription termination regions. There it mediates the recruitment of a specific, physiological binding partner, senataxin (SETX). Disruption of this complex led to R-loop-driven DNA damage at those loci as reflected by adjacent γ-H2AX accumulation and ssDNA breaks within the untranscribed strand of relevant R-loop structures. Genome-wide analysis revealed widespread BRCA1 binding enrichment at R-loop-rich termination regions (TRs) of actively transcribed genes. Strikingly, within some of these genes in BRCA1 null breast tumors, there are specific insertion/deletion mutations located close to R-loop-mediated BRCA1 binding sites within TRs. Thus, BRCA1/SETX complexes support a DNA repair mechanism that addresses R-loop-based DNA damage at transcriptional pause sites.

Highlights

Germline mutations of the breast cancer susceptibility gene 1 (BRCA1) significantly increase the risk of developing breast and ovarian cancers (Tutt and Ashworth, 2002)
Identification of BRCA1 as a Scaffolding Partner for SETX at the b-actin Transcription Termination Site To investigate whether BRCA1 is involved in R-loop-driven DNA damage responses, we first assessed the physiological relevance of a BRCA1 and SETX interaction, recently identified in our proteomic screens (Hill et al, 2014) and suggested by others (Becherel et al, 2013)
After depletion of either BRCA1 or SETX, we observed significantly increased signals of gH2AX largely restricted to the termination region (Figure 3A)

Summary

Introduction

Germline mutations of the breast cancer susceptibility gene 1 (BRCA1) significantly increase the risk of developing breast and ovarian cancers (Tutt and Ashworth, 2002). As part of several complexes, BRCA1 contributes to double-strand DNA break (DSB) repair by homologous recombination (HR), stalled fork repair, cell-cycle checkpoint activation, transcription regulation, heterochromatin maintenance, mitotic spindle formation, RNA splicing control, and estrogen metabolism (Gardini et al, 2014; Gorski et al, 2011; Mullan et al, 2006; Pathania et al, 2011; Savage et al, 2014a, 2014b; Venkitaraman, 2014; Zhu et al, 2011) Many of these BRCA1 properties and, in particular, those that protect genome integrity, probably contribute to its tumor suppressing function (Silver and Livingston, 2012; Tutt and Ashworth, 2002). Identifying new BRCA1 binding partners and their associated functions may yield valuable insights

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