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

Abstract

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...

Harmful DNA:RNA hybrids are formedin cisand in a Rad51-independent manner

“…This is consistent with the idea that it is the RNA produced in cis who invades the duplex DNA, a reaction that can be facilitated by increasing the negative supercoiling of DNA as well as by nicking the DNA template (Roy, Zhang, Lu, Hsieh, & Lieber, 2010). The evidence of DNA:RNA hybrid formation at breaks has matured in the last years (Cohen et al, 2018;D'Alessandro et al, 2018;L. Li et al, 2016;Ohle et al, 2016;Teng et al, 2018;Yasuhara et al, 2018) although the source and role of such hybrids remains still controversial Puget, Miller, & Legube, 2019).…”

The DNA damage response acts as a safeguard against harmful DNA–RNA hybrids of different origins

“…Also, the fact that RNase H overexpression had no effect on the number of breaks induced by ATM/CHK2 depletion ( Fig 7A) suggests that most breaks occurring in the absence of ATM/CHK2 are independent on DNA-RNA hybrids. Given the views and recent observations supporting that DNA breakage, whether single-or double-stranded, is a driving force for DNA-RNA hybrid formation [15,56,[64][65][66], the accumulation of DNA-RNA hybrids in ATM/CHK2-depleted cells might rather be a consequence of such unrepaired DSBs, which would not imply any additional consequences in fork progression.…”

Arginine methylation of the DDX 5 helicase RGG / RG motif by PRMT 5 regulates resolution of RNA:DNA hybrids

“…DDX1 also functions in tRNA, mRNA and miRNA processing, transport of RNAs from nucleus to cytoplasm, and AU‐rich element‐mediated mRNA decay in cytoplasm. Its function in R‐loop resolution facilitates homologous recombination repair (Li et al , ). DDX19 is well known for its functions in mRNA nuclear export at the nuclear pore.…”

“…Senataxin is reported to function with the 5′–3′ exonuclease XRN2 to resolve a subset of R‐loops at transcription termination sites of actively transcribed genes (Skourti‐Stathaki et al , ; Morales et al , ; Aymard et al , ). The DNA helicase RECQ5 and RNA helicases DDX1 (Li et al , , ; Ribeiro de Almeida et al , ), DDX19 (Hodroj et al , ), DDX21 (Song et al , ), DDX23 (Sridhara et al , ), and DHX9 (Cristini et al , ) were also found to be functionally involved in suppression of R‐loops. Topoisomerase I removes the negative supercoils behind RNA polymerases to prevent annealing of the nascent RNA with the DNA template and suppresses R‐loop formation (Tuduri et al , ).…”