DDX5 resolves R-loops at DNA double-strand breaks to promote DNA repair and avoid chromosomal deletions

Abstract

R-loops are three-stranded structures consisting of a DNA/RNA hybrid and a displaced DNA strand. The regulatory factors required to process this fundamental genetic structure near double-strand DNA breaks (DSBs) are not well understood. We previously reported that cellular depletion of the ATP-dependent DEAD box RNA helicase DDX5 increases R-loops genome-wide causing genomic instability. In this study, we define a pivotal role for DDX5 in clearing R-loops at or near DSBs enabling proper DNA repair to avoid aberrations such as chromosomal deletions. Remarkably, using the non-homologous end joining reporter gene (EJ5-GFP), we show that DDX5-deficient U2OS cells exhibited asymmetric end deletions on the side of the DSBs where there is overlap with a transcribed gene. Cross-linking and immunoprecipitation showed that DDX5 bound RNA transcripts near DSBs and required its helicase domain and the presence of DDX5 near DSBs was also shown by chromatin immunoprecipitation. DDX5 was excluded from DSBs in a transcription- and ATM activation-dependent manner. Using DNA/RNA immunoprecipitation, we show DDX5-deficient cells had increased R-loops near DSBs. Finally, DDX5 deficiency led to delayed exonuclease 1 and replication protein A recruitment to laser irradiation-induced DNA damage sites, resulting in homologous recombination repair defects. Our findings define a role for DDX5 in facilitating the clearance of RNA transcripts overlapping DSBs to ensure proper DNA repair.

BRCA2 promotes DNA‐RNA hybrid resolution by DDX5 helicase at DNA breaks to facilitate their repair‡

“…Importantly, we find that DDX5‐depleted cells exhibit an enrichment of DNA‐RNA hybrids at γH2AX positive regions suggesting DNA‐RNA hybrids accumulating particularly at DSBs in these cells. These results are consistent with a recent report that appeared while revising this manuscript (Yu et al , 2020).…”

“…This “anti‐stripe” pattern already reported before for other RBPs and DEAD‐box proteins (Chou et al , 2010; Adamson et al , 2012; Britton et al , 2014) started within the first 2 min post‐irradiation and reached 25% of the cells at 10 min post‐irradiation, whereas the rest of the cells showed DDX5 pan‐nuclear staining. While revising this manuscript, another report has also shown exclusion of GFP‐DDX5 from laser‐induced DNA damage using a different system (Yu et al , 2020). Interestingly, depletion of BRCA2 resulted in a widespread “anti‐stripe” pattern of DDX5‐GFP in the cell population reaching 63% of the cells at 6 min (Fig 4A).…”

New Faces of old Friends: Emerging new Roles of RNA-Binding Proteins in the DNA Double-Strand Break Response

“…Interestingly, upon DSB recruitment, CIRBP is poly (ADPribosyl)ated and then rapidly excluded again from the damaged chromatin (Chen et al, 2018). Similar association and dissociation dynamics have been observed for other RBPs as well (Salton et al, 2010;Ha et al, 2011;Adamson et al, 2012;Beli et al, 2012;Polo et al, 2012;Britton et al, 2014;Altmeyer et al, 2015;Yu et al, 2020;Sessa et al, 2021). Another RBP that is recruited to damaged chromatin upon DSB formation and that appears to directly regulate DSB signaling is WRAP53 (WD40encoding RNA antisense to p53, also known as TCAB1).…”

“…As such, it prevents R-loop formation by avoiding excessive RNAP IImediated transcription at DSB sites (Schwartz et al, 2012;Kwon et al, 2013;Hill et al, 2016). DDX5, a DEAD box-containing RNA helicase like DDX1, also unwinds RNA:DNA hybrids and R-loops at DSB sites (Xing et al, 2017;Mersaoui et al, 2019;Yu et al, 2020;Sessa et al, 2021). It binds to RNA that is transcribed in cis to a DSB to remove RNA-associated impediments and enable HR-directed repair (Yu et al, 2020;Sessa et al, 2021).…”

“…DDX5, a DEAD box-containing RNA helicase like DDX1, also unwinds RNA:DNA hybrids and R-loops at DSB sites (Xing et al, 2017;Mersaoui et al, 2019;Yu et al, 2020;Sessa et al, 2021). It binds to RNA that is transcribed in cis to a DSB to remove RNA-associated impediments and enable HR-directed repair (Yu et al, 2020;Sessa et al, 2021). Accordingly, loss of DDX5 results in the accumulation of polarized end deletions that specifically occur on those sides of DSBs that are actively transcribed and as such generate RNA species that can hinder the HR machinery (Yu et al, 2020).…”