DNA damage triggers SAF-A and RNA biogenesis factors exclusion from chromatin coupled to R-loops removal

Britton, Sébastien; Dernoncourt, Emma; Delteil, Christine; Froment, Carine; Burlet‐Schiltz, Odile; Salles, Bernard; Frit, Philippe; Calsou, Patrick

doi:10.1093/nar/gku601

Cited by 166 publications

(181 citation statements)

References 85 publications

(101 reference statements)

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“…The ChIP signal was the same at R-loops from wildtype and catalytically dead RNase H1. If RNase H1 were active on most hybrids where it was bound, one might expect it to degrade the hybrids and interact transiently, whereas the catalytically dead RNase H1, being unable to degrade the hybrids, would have a prolonged interaction and generate a higher ChIP signal (17,18). However, this was not the case.…”

Section: Discussionmentioning

confidence: 99%

Differential roles of the RNases H in preventing chromosome instability

Zimmer

Koshland

2016

Proc. Natl. Acad. Sci. U.S.A.

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DNA:RNA hybrids can lead to DNA damage and genome instability. This damage can be prevented by degradation of the RNA in the hybrid by two evolutionarily conserved enzymes, RNase H1 and H2. Indeed, RNase H-deficient cells have increased chromosomal rearrangements. However, the quantitative and spatial contributions of the individual enzymes to hybrid removal have been unclear. Additionally, RNase H2 can remove single ribonucleotides misincorporated into DNA during replication. The relative contribution of DNA:RNA hybrids and misincorporated ribonucleotides to chromosome instability also was uncertain. To address these issues, we studied the frequency and location of loss-ofheterozygosity (LOH) events on chromosome III in Saccharomyces cerevisiae strains that were defective for RNase H1, H2, or both. We showed that RNase H2 plays the major role in preventing chromosome III instability through its hybrid-removal activity. Furthermore, RNase H2 acts pervasively at many hybrids along the chromosome. In contrast, RNase H1 acts to prevent LOH within a small region of chromosome III where the instability is dependent upon two hybrid-prone sequences. This restriction of RNase H1 activity to a subset of hybrids is not the result of its constrained localization, because we found it at hybrids genome-wide. This result suggests that the genome-protection activity of RNase H1 is regulated at a step after hybrid recognition. The global function of RNase H2 and the region-specific function of RNase H1 provide insight into why these enzymes with overlapping hybrid-removal activities have been conserved throughout evolution.RNase H | R-loops | DNA:RNA hybrids | chromosome instability | genome instability P reventing chromosome instability is an essential process for maintaining genetic information. A source of chromosome instability is the accumulation of R-loops, which form when an RNA molecule hybridizes with a portion of genomic DNA, creating a DNA:RNA hybrid and a displaced single-stranded DNA (reviewed in ref. 1). One mechanism to prevent hybridmediated damage involves RNase H1 and RNase H2, two endogenous enzymes, conserved from bacteria to humans, that can degrade the RNA in R-loops (reviewed in ref.2). RNase H2 also functions in the removal of single ribonucleotides that are inappropriately incorporated into DNA by DNA polymerases during replication. Why RNase H1 and H2, which appear to have overlapping functions, remain highly conserved across many branches of life has been an outstanding question. Two areas of inquiry that will help address this conundrum are (i) does one of these RNases carry the major burden of preventing spontaneous R-loop-mediated chromosome instability, and, if so which, and (ii) do RNase H1 and H2 protect the same or different regions of the genome from R-loop-mediated damage?Whether RNase H1 and H2 contribute differentially to protecting against hybrid-mediated genome instability has been controversial. Studies of the inactivation of RNase H1 in yeast have shown little effect on chromosome insta...

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Section: Discussionmentioning

confidence: 99%

Differential roles of the RNases H in preventing chromosome instability

Zimmer

Koshland

2016

Proc. Natl. Acad. Sci. U.S.A.

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“…A growing body of evidence suggests that cotranscriptional R-loops contribute to DSB formation, possibly due to collisions between the replication and transcription machineries at the unresolved R-loops (13)(14)(15). In addition, Rloops as detected by RNase H recruitment have also been observed at micro-IR-induced DNA lesions immediately following DNA damage (20).…”

Section: Discussionmentioning

confidence: 99%

“…Second, although others have shown that R-loops and R-loop-induced phenotypes can be suppressed by overexpression of RNase H (17,18,54), RNase H overexpression did not compensate for DDX1 depletion as measured by the DR-GFP reporter assay. Third, recruitment of RNase H and other R-loop-associated factors to DNA lesions induced by micro-IR is rapid but transient (recruitment occurs within minutes, with factors then dissociating from the DNA lesions) (20), whereas DDX1 accumulation at DSBs follows much slower kinetics (peaks at 1 h, with no dissociation detected for hours), suggesting that RNase H and DDX1 either play different roles or act at different stages in resolving RNA-DNA structures during the DSB repair process. Fourth, senataxin, an RNA helicase that plays a critical role in resolving R-loops in vivo (54,64), did not coimmunoprecipitate with DDX1 in either the presence or absence of IR-induced DNA damage.…”

Section: Discussionmentioning

confidence: 99%

“…6E) further implicates RNA-DNA duplexes in DDX1-mediated aspects of DSB repair by HR. R-loops consisting of RNA-DNA duplexes have been implicated in DSB formation and the ensuing cellular response (18)(19)(20). We therefore examined whether DDX1 colocalizes with two known markers associated with R-loops: the S9.6 antibody, which specifically recognizes RNA-DNA hybrids (53), and senataxin, an RNA helicase that resolves aberrant R-loops at transcriptionally active chromatin to facilitate proper transcription termination (54).…”

Section: Ddx1 Contributes To Dsb Repair and Cell Survival Post-irmentioning

confidence: 99%

“…Together, these studies suggest that deregulated RNA-DNA hybrid formation during transcription can result in the accumulation of DSBs and genome instability. In addition to triggering DSB formation, recent work by Britton et al (20) suggests that removal of transcription-coupled R-loops in the vicinity of DNA damage generated by microirradiation (micro-IR) is an integral part of the cellular response to DSBs. However, it is not known whether RNA molecules bind DNA at the site of DSBs during repair and, if so, whether RNA-DNA duplexes can impact repair pathway choice and/or efficiency.…”

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DEAD Box 1 Facilitates Removal of RNA and Homologous Recombination at DNA Double-Strand Breaks

Germain

Poon

et al. 2016

Molecular and Cellular Biology

133

138

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Although RNA and RNA-binding proteins have been linked to double-strand breaks (DSBs), little is known regarding their roles in the cellular response to DSBs and, if any, in the repair process. Here, we provide direct evidence for the presence of RNA-DNA hybrids at DSBs and suggest that binding of RNA to DNA at DSBs may impact repair efficiency. Our data indicate that the RNAunwinding protein DEAD box 1 (DDX1) is required for efficient DSB repair and cell survival after ionizing radiation (IR), with depletion of DDX1 resulting in reduced DSB repair by homologous recombination (HR). While DDX1 is not essential for end resection, a key step in homology-directed DSB repair, DDX1 is required for maintenance of the single-stranded DNA once generated by end resection. We show that transcription deregulation has a significant effect on DSB repair by HR in DDX1-depleted cells and that RNA-DNA duplexes are elevated at DSBs in DDX1-depleted cells. Based on our combined data, we propose a role for DDX1 in resolving RNA-DNA structures that accumulate at DSBs located at sites of active transcription. Our findings point to a previously uncharacterized requirement for clearing RNA at DSBs for efficient repair by HR. DEAD box proteins are a family of putative RNA helicases that function by altering RNA secondary structure. This protein family has been implicated in all aspects of RNA metabolism. The DEAD box 1 gene (DDX1) is a widely expressed gene that is misexpressed in a number of cancers, including retinoblastoma, neuroblastoma, and breast cancer (1, 2). Knockout of DDX1 leads to early embryonic lethality in mice and severely reduced fertility in flies (3, 4). DDX1 is involved in the transport of RNAs from the nucleus to the cytoplasm and regulates cytoplasmic localization of the splicing-regulatory protein KSRP (5). In neurons, DDX1 resides in RNA-transporting granules, cytoplasmic organelles that regulate the localization and expression of target mRNAs (6, 7). DDX1 has also been identified as a core subunit of the human tRNA ligase complex which is essential for tRNA splicing (8).In addition to its roles in RNA metabolism, DDX1 has been implicated in the cellular response to DNA double-strand breaks (DSBs). Upon treatment of cells with ionizing radiation (IR), DDX1 rapidly accumulates at a subset of DNA DSBs (ϳ30%), where it forms IR-induced foci that colocalize with ␥-H2AX, a marker for DSBs (9). DDX1 coimmunoprecipitates with the MRN (MRE11-RAD50-NBS1) complex, the early sensor of DNA DSBs, and ATM (ataxia telangiectasia mutated) protein, the key transducer of the signaling cascade in response to DSBs (10, 11). DSBs induce DDX1 phosphorylation in an ATM-dependent manner. Notably, IR-induced DDX1 foci are lost when cells are treated with RNase H, an enzyme that specifically digests RNA from RNA-DNA hybrids (9). These results suggest that RNA-DNA double-stranded structures are required for the presence and/or retention of DDX1 at DSBs. In line with this observation, biochemical analysis has shown that DDX1 can unwind both...

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Looping forward: exploring R‐loop processing and therapeutic potential

Stratigi,

Siametis,

Garinis

2024

FEBS Letters

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Recently, there has been increasing interest in the complex relationship between transcription and genome stability, with specific attention directed toward the physiological significance of molecular structures known as R‐loops. These structures arise when an RNA strand invades into the DNA duplex, and their formation is involved in a wide range of regulatory functions affecting gene expression, DNA repair processes or cell homeostasis. The persistent presence of R‐loops, if not effectively removed, contributes to genome instability, underscoring the significance of the factors responsible for their resolution and modification. In this review, we provide a comprehensive overview of how R‐loop processing can drive either a beneficial or a harmful outcome. Additionally, we explore the potential for manipulating such structures to devise rationalized therapeutic strategies targeting the aberrant accumulation of R‐loops.

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DNA damage triggers SAF-A and RNA biogenesis factors exclusion from chromatin coupled to R-loops removal

Cited by 166 publications

References 85 publications

Differential roles of the RNases H in preventing chromosome instability

Differential roles of the RNases H in preventing chromosome instability

DEAD Box 1 Facilitates Removal of RNA and Homologous Recombination at DNA Double-Strand Breaks

Looping forward: exploring R‐loop processing and therapeutic potential

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