RNA–DNA hybrids are generated during transcription, DNA replication and DNA repair and are crucial intermediates in these processes. When RNA–DNA hybrids are stably formed in double-stranded DNA, they displace one of the DNA strands and give rise to a three-stranded structure called an R-loop. R-loops are widespread in the genome and are enriched at active genes. R-loops have important roles in regulating gene expression and chromatin structure, but they also pose a threat to genomic stability, especially during DNA replication. To keep the genome stable, cells have evolved a slew of mechanisms to prevent aberrant R-loop accumulation. Although R-loops can cause DNA damage, they are also induced by DNA damage and act as key intermediates in DNA repair such as in transcription-coupled repair and RNA-templated DNA break repair. When the regulation of R-loops goes awry, pathological R-loops accumulate, which contributes to diseases such as neurodegeneration and cancer. In this Review, we discuss the current understanding of the sources of R-loops and RNA–DNA hybrids, mechanisms that suppress and resolve these structures, the impact of these structures on DNA repair and genome stability, and opportunities to therapeutically target pathological R-loops.
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Crossley, M. P., Bocek, M. & Cimprich, K. A. R-loops as cellular regulators and genomic threats. Mol. Cell 73, 398–411 (2019).
Garcia-Muse, T. & Aguilera, A. R loops: from physiological to pathological roles. Cell 179, 604–618 (2019).
Niehrs, C. & Luke, B. Regulatory R-loops as facilitators of gene expression and genome stability. Nat. Rev. Mol. Cell Biol. 21, 167–178 (2020).
Marnef, A. & Legube, G. R-loops as Janus-faced modulators of DNA repair. Nat. Cell Biol. 23, 305–313 (2021).
Stodola, J. L. & Burgers, P. M. Mechanism of lagging-strand DNA replication in eukaryotes. Adv. Exp. Med. Biol. 1042, 117–133 (2017).
Sanz, L. A. et al. Prevalent, dynamic, and conserved R-loop structures associate with specific epigenomic signatures in mammals. Mol. Cell 63, 167–178 (2016).
Chen, J. Y., Zhang, X., Fu, X. D. & Chen, L. R-ChIP for genome-wide mapping of R-loops by using catalytically inactive RNASEH1. Nat. Protoc. 14, 1661–1685 (2019).
Chiang, H. C. et al. BRCA1-associated R-loop affects transcription and differentiation in breast luminal epithelial cells. Nucleic Acids Res. 47, 5086–5099 (2019).
Gorthi, A. et al. EWS-FLI1 increases transcription to cause R-loops and block BRCA1 repair in Ewing sarcoma. Nature 555, 387–391 (2018).
Patel, P. S. et al. RNF168 regulates R-loop resolution and genomic stability in BRCA1/2-deficient tumors. J. Clin. Invest. https://doi.org/10.1172/JCI140105 (2021).
Jones, S. E. et al. ATR is a therapeutic target in synovial sarcoma. Cancer Res. 77, 7014–7026 (2017).
Ujvari, A. & Luse, D. S. RNA emerging from the active site of RNA polymerase II interacts with the Rpb7 subunit. Nat. Struct. Mol. Biol. 13, 49–54 (2006).
Ginno, P. A., Lott, P. L., Christensen, H. C., Korf, I. & Chedin, F. R-loop formation is a distinctive characteristic of unmethylated human CpG island promoters. Mol. Cell 45, 814–825 (2012).
Skourti-Stathaki, K., Proudfoot, N. J. & Gromak, N. Human senataxin resolves RNA/DNA hybrids formed at transcriptional pause sites to promote Xrn2-dependent termination. Mol. Cell 42, 794–805 (2011).
Skourti-Stathaki, K., Kamieniarz-Gdula, K. & Proudfoot, N. J. R-loops induce repressive chromatin marks over mammalian gene terminators. Nature 516, 436–439 (2014).
Boque-Sastre, R. et al. Head-to-head antisense transcription and R-loop formation promotes transcriptional activation. Proc. Natl Acad. Sci. USA 112, 5785–5790 (2015).
Yu, K., Chedin, F., Hsieh, C. L., Wilson, T. E. & Lieber, M. R. R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells. Nat. Immunol. 4, 442–451 (2003).
Stork, C. T. et al. Co-transcriptional R-loops are the main cause of estrogen-induced DNA damage. eLife 5, e17548 (2016).
Kotsantis, P. et al. Increased global transcription activity as a mechanism of replication stress in cancer. Nat. Commun. 7, 13087 (2016).
Chen, L. et al. The Augmented R-loop is a unifying mechanism for myelodysplastic syndromes induced by high-risk splicing factor mutations. Mol. Cell 69, 412–425.e6 (2018).
El Hage, A., French, S. L., Beyer, A. L. & Tollervey, D. Loss of Topoisomerase I leads to R-loop-mediated transcriptional blocks during ribosomal RNA synthesis. Genes Dev. 24, 1546–1558 (2010).
Abraham, K. J. et al. Nucleolar RNA polymerase II drives ribosome biogenesis. Nature 585, 298–302 (2020).
El Hage, A., Webb, S., Kerr, A. & Tollervey, D. Genome-wide distribution of RNA-DNA hybrids identifies RNase H targets in tRNA genes, retrotransposons and mitochondria. PLoS Genet. 10, e1004716 (2014).
Azzalin, C. M., Reichenbach, P., Khoriauli, L., Giulotto, E. & Lingner, J. Telomeric repeat containing RNA and RNA surveillance factors at mammalian chromosome ends. Science 318, 798–801 (2007).
Schoeftner, S. & Blasco, M. A. Developmentally regulated transcription of mammalian telomeres by DNA-dependent RNA polymerase II. Nat. Cell Biol. 10, 228–236 (2008).
Luke, B. & Lingner, J. TERRA: telomeric repeat-containing RNA. EMBO J. 28, 2503–2510 (2009).
Arora, R. et al. RNaseH1 regulates TERRA-telomeric DNA hybrids and telomere maintenance in ALT tumour cells. Nat. Commun. 5, 5220 (2014).
Feretzaki, M. et al. RAD51-dependent recruitment of TERRA lncRNA to telomeres through R-loops. Nature 587, 303–308 (2020).
Talbert, P. B. & Henikoff, S. Transcribing centromeres: noncoding RNAs and kinetochore assembly. Trends Genet. 34, 587–599 (2018).
Bobkov, G. O. M., Gilbert, N. & Heun, P. Centromere transcription allows CENP-A to transit from chromatin association to stable incorporation. J. Cell Biol. 217, 1957–1972 (2018).
McNulty, S. M., Sullivan, L. L. & Sullivan, B. A. Human centromeres produce chromosome-specific and array-specific alpha satellite transcripts that are complexed with CENP-A and CENP-C. Dev. Cell 42, 226–240.e6 (2017).
Kabeche, L., Nguyen, H. D., Buisson, R. & Zou, L. A mitosis-specific and R loop-driven ATR pathway promotes faithful chromosome segregation. Science 359, 108–114 (2018).
Giunta, S. et al. CENP-A chromatin prevents replication stress at centromeres to avoid structural aneuploidy. Proc. Natl Acad. Sci. USA 118, e2015634118 (2021).
Smith, D. J. & Whitehouse, I. Intrinsic coupling of lagging-strand synthesis to chromatin assembly. Nature 483, 434–438 (2012).
Gloor, J. W., Balakrishnan, L., Campbell, J. L. & Bambara, R. A. Biochemical analyses indicate that binding and cleavage specificities define the ordered processing of human Okazaki fragments by Dna2 and FEN1. Nucleic Acids Res. 40, 6774–6786 (2012).
Bianchi, J. et al. PrimPol bypasses UV photoproducts during eukaryotic chromosomal DNA replication. Mol. Cell 52, 566–573 (2013).
Garcia-Gomez, S. et al. PrimPol, an archaic primase/polymerase operating in human cells. Mol. Cell 52, 541–553 (2013).
Reijns, M. A. et al. Enzymatic removal of ribonucleotides from DNA is essential for mammalian genome integrity and development. Cell 149, 1008–1022 (2012).
Sparks, J. L. et al. RNase H2-initiated ribonucleotide excision repair. Mol. Cell 47, 980–986 (2012).
Wei, W. et al. A role for small RNAs in DNA double-strand break repair. Cell 149, 101–112 (2012).
Francia, S. et al. Site-specific DICER and DROSHA RNA products control the DNA-damage response. Nature 488, 231–235 (2012).
Pessina, F. et al. Functional transcription promoters at DNA double-strand breaks mediate RNA-driven phase separation of damage-response factors. Nat. Cell Biol. 21, 1286–1299 (2019).
Sharma, S. et al. MRE11-RAD50-NBS1 complex is sufficient to promote transcription by RNA polymerase II at double-strand breaks by melting DNA ends. Cell Rep. 34, 108565 (2021).
Liu, S. et al. RNA polymerase III is required for the repair of DNA double-strand breaks by homologous recombination. Cell 184, 1314–1329.e10 (2021).
Cohen, S. et al. Senataxin resolves RNA:DNA hybrids forming at DNA double-strand breaks to prevent translocations. Nat. Commun. 9, 533 (2018).
Teng, Y. et al. ROS-induced R loops trigger a transcription-coupled but BRCA1/2-independent homologous recombination pathway through CSB. Nat. Commun. 9, 4115 (2018).
Holt, I. J. The mitochondrial R-loop. Nucleic Acids Res. 47, 5480–5489 (2019).
Falkenberg, M. Mitochondrial DNA replication in mammalian cells: overview of the pathway. Essays Biochem. 62, 287–296 (2018).
Lima, W. F. et al. Viable RNaseH1 knockout mice show RNaseH1 is essential for R loop processing, mitochondrial and liver function. Nucleic Acids Res. 44, 5299–5312 (2016).
Silva, S., Camino, L. P. & Aguilera, A. Human mitochondrial degradosome prevents harmful mitochondrial R loops and mitochondrial genome instability. Proc. Natl Acad. Sci. USA 115, 11024–11029 (2018).
Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278 (2014).
Doudna, J. A. & Charpentier, E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346, 1258096 (2014).
Jiang, F. et al. Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage. Science 351, 867–871 (2016).
Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019).
Gaudelli, N. M. et al. Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017).
Xiao, Y. et al. Structure basis for directional r-loop formation and substrate handover mechanisms in type I CRISPR-Cas System. Cell 170, e11 (2017).
Zhang, B. et al. Mechanistic insights into the R-loop formation and cleavage in CRISPR-Cas12i1. Nat. Commun. 12, 3476 (2021).
Gan, W. et al. R-loop-mediated genomic instability is caused by impairment of replication fork progression. Genes Dev. 25, 2041–2056 (2011).
Hamperl, S., Bocek, M. J., Saldivar, J. C., Swigut, T. & Cimprich, K. A. Transcription-replication conflict orientation modulates R-loop levels and activates distinct DNA damage responses. Cell 170, 774–786.e19 (2017).
Lang, K. S. et al. Replication-transcription conflicts generate R-loops that orchestrate bacterial stress survival and pathogenesis. Cell 170, 787–799.e18 (2017).
Matos, D. A. et al. ATR protects the genome against R Loops through a MUS81-triggered feedback loop. Mol. Cell 77, 514–527.e4 (2020).
Promonet, A. et al. Topoisomerase 1 prevents replication stress at R-loop-enriched transcription termination sites. Nat. Commun. 11, 3940 (2020).
Lee, W. T. C. et al. Single-molecule imaging reveals replication fork coupled formation of G-quadruplex structures hinders local replication stress signaling. Nat. Commun. 12, 2525 (2021).
Kotsantis, P. et al. RTEL1 Regulates G4/R-loops to avert replication-transcription collisions. Cell Rep. 33, 108546 (2020).
Drosopoulos, W. C., Kosiyatrakul, S. T. & Schildkraut, C. L. BLM helicase facilitates telomere replication during leading strand synthesis of telomeres. J. Cell Biol. 210, 191–208 (2015).
Sarkies, P. et al. FANCJ coordinates two pathways that maintain epigenetic stability at G-quadruplex DNA. Nucleic Acids Res. 40, 1485–1498 (2012).
Neil, A. J., Liang, M. U., Khristich, A. N., Shah, K. A. & Mirkin, S. M. RNA-DNA hybrids promote the expansion of Friedreich’s ataxia (GAA)n repeats via break-induced replication. Nucleic Acids Res. 46, 3487–3497 (2018).
Loomis, E. W., Sanz, L. A., Chedin, F. & Hagerman, P. J. Transcription-associated R-loop formation across the human FMR1 CGG-repeat region. PLoS Genet. 10, e1004294 (2014).
Castellano-Pozo, M. et al. R loops are linked to histone H3 S10 phosphorylation and chromatin condensation. Mol. Cell 52, 583–590 (2013).
Bayona-Feliu, A., Casas-Lamesa, A., Reina, O., Bernues, J. & Azorin, F. Linker histone H1 prevents R-loop accumulation and genome instability in heterochromatin. Nat. Commun. 8, 283 (2017).
Sollier, J. et al. Transcription-coupled nucleotide excision repair factors promote R-loop-induced genome instability. Mol. Cell 56, 777–785 (2014).
Cristini, A. et al. Dual processing of R-loops and topoisomerase I induces transcription-dependent DNA double-strand breaks. Cell Rep. 28, e3166 (2019).
Buisson, R., Lawrence, M. S., Benes, C. H. & Zou, L. APOBEC3A and APOBEC3B activities render cancer cells susceptible to ATR inhibition. Cancer Res. 77, 4567–4578 (2017).
Cerritelli, S. M. et al. Failure to produce mitochondrial DNA results in embryonic lethality in Rnaseh1 null mice. Mol. Cell 11, 807–815 (2003).
Bubeck, D. et al. PCNA directs type 2 RNase H activity on DNA replication and repair substrates. Nucleic Acids Res. 39, 3652–3666 (2011).
Kind, B. et al. Altered spatio-temporal dynamics of RNase H2 complex assembly at replication and repair sites in Aicardi-Goutieres syndrome. Hum. Mol. Genet. 23, 5950–5960 (2014).
Lockhart, A. et al. RNase H1 and H2 are differentially regulated to process RNA-DNA hybrids. Cell Rep. 29, 2890–2900.e5 (2019).
Zatreanu, D. et al. Elongation factor TFIIS prevents transcription stress and R-loop accumulation to maintain genome stability. Mol. Cell 76, 57–69.e9 (2019).
Li, X. & Manley, J. L. Inactivation of the SR protein splicing factor ASF/SF2 results in genomic instability. Cell 122, 365–378 (2005).
Chakraborty, P., Huang, J. T. J. & Hiom, K. DHX9 helicase promotes R-loop formation in cells with impaired RNA splicing. Nat. Commun. 9, 4346 (2018).
Sorrells, S. et al. Spliceosomal components protect embryonic neurons from R-loop-mediated DNA damage and apoptosis. Dis. Model. Mech. 11, dmm031583 (2018).
Puhringer, T. et al. Structure of the human core transcription-export complex reveals a hub for multivalent interactions. eLife 9, e61503 (2020).
Salas-Armenteros, I. et al. Human THO-Sin3A interaction reveals new mechanisms to prevent R-loops that cause genome instability. EMBO J. 36, 3532–3547 (2017).
Bhatia, V. et al. BRCA2 prevents R-loop accumulation and associates with TREX-2 mRNA export factor PCID2. Nature 511, 362–365 (2014).
Nojima, T. et al. Deregulated expression of mammalian lncRNA through Loss of SPT6 Induces R-loop formation, replication stress, and cellular senescence. Mol. Cell 72, 970–984.e7 (2018).
Pefanis, E. et al. RNA exosome-regulated long non-coding RNA transcription controls super-enhancer activity. Cell 161, 774–789 (2015).
Manzo, S. G. et al. DNA Topoisomerase I differentially modulates R-loops across the human genome. Genome Biol. 19, 100 (2018).
Hatchi, E. et al. BRCA1 recruitment to transcriptional pause sites is required for R-loop-driven DNA damage repair. Mol. Cell 57, 636–647 (2015).
Jurga, M., Abugable, A. A., Goldman, A. S. H. & El-Khamisy, S. F. USP11 controls R-loops by regulating senataxin proteostasis. Nat. Commun. 12, 5156 (2021).
Perez-Calero, C. et al. UAP56/DDX39B is a major cotranscriptional RNA-DNA helicase that unwinds harmful R loops genome-wide. Genes Dev. 34, 898–912 (2020).
Song, C., Hotz-Wagenblatt, A., Voit, R. & Grummt, I. SIRT7 and the DEAD-box helicase DDX21 cooperate to resolve genomic R loops and safeguard genome stability. Genes Dev. 31, 1370–1381 (2017).
Cristini, A., Groh, M., Kristiansen, M. S. & Gromak, N. RNA/DNA hybrid interactome identifies DXH9 as a molecular player in transcriptional termination and R-loop-associated DNA damage. Cell Rep. 23, 1891–1905 (2018).
Chakraborty, P. & Grosse, F. Human DHX9 helicase preferentially unwinds RNA-containing displacement loops (R-loops) and G-quadruplexes. DNA Repair 10, 654–665 (2011).
Rivosecchi, J. et al. Senataxin homologue Sen1 is required for efficient termination of RNA polymerase III transcription. EMBO J. 38, e101955 (2019).
Osmundson, J. S., Kumar, J., Yeung, R. & Smith, D. J. Pif1-family helicases cooperatively suppress widespread replication-fork arrest at tRNA genes. Nat. Struct. Mol. Biol. 24, 162–170 (2017).
Tran, P. L. T. et al. PIF1 family DNA helicases suppress R-loop mediated genome instability at tRNA genes. Nat. Commun. 8, 15025 (2017).
Zhang, X. et al. Attenuation of RNA polymerase II pausing mitigates BRCA1-associated R-loop accumulation and tumorigenesis. Nat. Commun. 8, 15908 (2017).
Shivji, M. K. K., Renaudin, X., Williams, C. H. & Venkitaraman, A. R. BRCA2 regulates transcription elongation by RNA polymerase II to prevent R-loop accumulation. Cell Rep. 22, 1031–1039 (2018).
Gomez-Gonzalez, B., Sessa, G., Carreira, A. & Aguilera, A. A new interaction between BRCA2 and DDX5 promotes the repair of DNA breaks at transcribed chromatin. Mol. Cell Oncol. 8, 1910474 (2021).
Bayona-Feliu, A., Barroso, S., Munoz, S. & Aguilera, A. The SWI/SNF chromatin remodeling complex helps resolve R-loop-mediated transcription-replication conflicts. Nat. Genet. 53, 1050–1063 (2021).
Madireddy, A. et al. FANCD2 facilitates replication through common fragile sites. Mol. Cell 64, 388–404 (2016).
Okamoto, Y. et al. SLFN11 promotes stalled fork degradation that underlies the phenotype in Fanconi anemia cells. Blood 137, 336–348 (2021).
Liang, Z. et al. Binding of FANCI-FANCD2 complex to RNA and R-loops stimulates robust FANCD2 monoubiquitination. Cell Rep. 26, 564–572.e5 (2019).
Nguyen, H. D. et al. Functions of replication protein A as a sensor of R loops and a regulator of RNaseH1. Mol. Cell 65, 832–847.e4 (2017).
Makharashvili, N. et al. Sae2/CtIP prevents R-loop accumulation in eukaryotic cells. eLife 7, e42733 (2018).
Pan, X. et al. FANCM suppresses DNA replication stress at ALT telomeres by disrupting TERRA R-loops. Sci. Rep. 9, 19110 (2019).
Silva, B. et al. FANCM limits ALT activity by restricting telomeric replication stress induced by deregulated BLM and R-loops. Nat. Commun. 10, 2253 (2019).
Tan, J., Wang, X., Phoon, L., Yang, H. & Lan, L. Resolution of ROS-induced G-quadruplexes and R-loops at transcriptionally active sites is dependent on BLM helicase. FEBS Lett. 594, 1359–1367 (2020).
Pan, X., Ahmed, N., Kong, J. & Zhang, D. Breaking the end: target the replication stress response at the ALT telomeres for cancer therapy. Mol. Cell Oncol. 4, e1360978 (2017).
Alzu, A. et al. Senataxin associates with replication forks to protect fork integrity across RNA-polymerase-II-transcribed genes. Cell 151, 835–846 (2012).
Kim, S. et al. ATAD5 restricts R-loop formation through PCNA unloading and RNA helicase maintenance at the replication fork. Nucleic Acids Res. 48, 7218–7238 (2020).
Hodroj, D. et al. An ATR-dependent function for the Ddx19 RNA helicase in nuclear R-loop metabolism. EMBO J. 36, 1182–1198 (2017).
Chang, E. Y. et al. MRE11-RAD50-NBS1 promotes Fanconi anemia R-loop suppression at transcription-replication conflicts. Nat. Commun. 10, 4265 (2019).
Barroso, S. et al. The DNA damage response acts as a safeguard against harmful DNA-RNA hybrids of different origins. EMBO Rep. 20, e47250 (2019).
Chappidi, N. et al. Fork cleavage-religation cycle and active transcription mediate replication restart after fork stalling at co-transcriptional R-loops. Mol. Cell 77, 528–541.e8 (2020).
Svikovic, S. et al. R-loop formation during S phase is restricted by PrimPol-mediated repriming. EMBO J. 38, e99793 (2019).
Tsai, S. et al. ARID1A regulates R-loop associated DNA replication stress. PLoS Genet. 17, e1009238 (2021).
Prendergast, L. et al. Resolution of R-loops by INO80 promotes DNA replication and maintains cancer cell proliferation and viability. Nat. Commun. 11, 4534 (2020).
Herrera-Moyano, E., Mergui, X., Garcia-Rubio, M. L., Barroso, S. & Aguilera, A. The yeast and human FACT chromatin-reorganizing complexes solve R-loop-mediated transcription-replication conflicts. Genes Dev. 28, 735–748 (2014).
Sanchez, A. et al. Transcription-replication conflicts as a source of common fragile site instability caused by BMI1-RNF2 deficiency. PLoS Genet. 16, e1008524 (2020).
Klusmann, I. et al. Chromatin modifiers Mdm2 and RNF2 prevent RNA:DNA hybrids that impair DNA replication. Proc. Natl Acad. Sci. USA 115, E11311 (2018).
Singh, D. K. et al. MOF suppresses replication stress and contributes to resolution of stalled replication forks. Mol. Cell. Biol. 38, e00484-17 (2018).
Kim, J. J. et al. Systematic bromodomain protein screens identify homologous recombination and R-loop suppression pathways involved in genome integrity. Genes Dev. 33, 1751–1774 (2019).
Lam, F. C. et al. BRD4 prevents the accumulation of R-loops and protects against transcription–replication collision events and DNA damage. Nat. Commun. 11, 4083 (2020).
Edwards, D. S. et al. BRD4 prevents R-loop formation and transcription-replication conflicts by ensuring efficient transcription elongation. Cell Rep. 32, 108166 (2020).
Mognato, M., Burdak-Rothkamm, S. & Rothkamm, K. Interplay between DNA replication stress, chromatin dynamics and DNA-damage response for the maintenance of genome stability. Mutat. Res. 787, 108346 (2021).
Gao, M. et al. Ago2 facilitates Rad51 recruitment and DNA double-strand break repair by homologous recombination. Cell Res. 24, 532–541 (2014).
Michelini, F. et al. Damage-induced lncRNAs control the DNA damage response through interaction with DDRNAs at individual double-strand breaks. Nat. Cell Biol. 19, 1400–1411 (2017).
D’Alessandro, G. et al. BRCA2 controls DNA:RNA hybrid level at DSBs by mediating RNase H2 recruitment. Nat. Commun. 9, 5376 (2018).
Keskin, H. et al. Transcript-RNA-templated DNA recombination and repair. Nature 515, 436–439 (2014).
Yasuhara, T. et al. Human Rad52 promotes XPG-mediated R-loop processing to initiate transcription-associated homologous recombination repair. Cell 175, 558–570.e11 (2018).
Ohle, C. et al. Transient RNA-DNA hybrids are required for efficient double-strand break repair. Cell 167, 1001–1013.e7 (2016).
Chakraborty, P. & Hiom, K. DHX9-dependent recruitment of BRCA1 to RNA promotes DNA end resection in homologous recombination. Nat. Commun. 12, 4126 (2021).
Mazina, O. M., Keskin, H., Hanamshet, K., Storici, F. & Mazin, A. V. Rad52 Inverse strand exchange drives RNA-templated DNA double-strand break repair. Mol. Cell 67, 19–29.e3 (2017).
Wei, L. et al. DNA damage during the G0/G1 phase triggers RNA-templated, Cockayne syndrome B-dependent homologous recombination. Proc. Natl Acad. Sci. USA 112, E3495–E3504 (2015).
McDevitt, S., Rusanov, T., Kent, T., Chandramouly, G. & Pomerantz, R. T. How RNA transcripts coordinate DNA recombination and repair. Nat. Commun. 9, 1091 (2018).
Chandramouly, G. et al. Poltheta reverse transcribes RNA and promotes RNA-templated DNA repair. Sci. Adv. 7, eabf1771 (2021).
Ouyang, J. et al. RNA transcripts stimulate homologous recombination by forming DR-loops. Nature 594, 283–288 (2021).
Liang, F. et al. Promotion of RAD51-mediated homologous DNA pairing by the RAD51AP1-UAF1 complex. Cell Rep. 15, 2118–2126 (2016).
Kang, H. J. et al. TonEBP recognizes R-loops and initiates m6A RNA methylation for R-loop resolution. Nucleic Acids Res. 49, 269–284 (2021).
Abakir, A. et al. N(6)-methyladenosine regulates the stability of RNA:DNA hybrids in human cells. Nat. Genet. 52, 48–55 (2020).
Yang, X. et al. m(6)A promotes R-loop formation to facilitate transcription termination. Cell Res. 29, 1035–1038 (2019).
Xiang, Y. et al. RNA m(6)A methylation regulates the ultraviolet-induced DNA damage response. Nature 543, 573–576 (2017).
Zhang, C. et al. METTL3 and N6-methyladenosine promote homologous recombination-mediated repair of DSBs by modulating DNA-RNA hybrid accumulation. Mol. Cell 79, 425–442.e7 (2020).
Chen, H. et al. m(5)C modification of mRNA serves a DNA damage code to promote homologous recombination. Nat. Commun. 11, 2834 (2020).
Jimeno, S. et al. ADAR-mediated RNA editing of DNA:RNA hybrids is required for DNA double strand break repair. Nat. Commun. 12, 5512 (2021).
Graf, M. et al. Telomere length determines TERRA and R-loop regulation through the cell cycle. Cell 170, 72–85.e14 (2017).
Silva, B., Arora, R., Bione, S. & Azzalin, C. M. TERRA transcription destabilizes telomere integrity to initiate break-induced replication in human ALT cells. Nat. Commun. 12, 3760 (2021).
Zhang, J. M., Yadav, T., Ouyang, J., Lan, L. & Zou, L. Alternative lengthening of telomeres through two distinct break-induced replication pathways. Cell Rep. 26, 955–968.e3 (2019).
Tan, J. et al. An R-loop-initiated CSB-RAD52-POLD3 pathway suppresses ROS-induced telomeric DNA breaks. Nucleic Acids Res. 48, 1285–1300 (2020).
Hatchi, E. et al. BRCA1 and RNAi factors promote repair mediated by small RNAs and PALB2-RAD52. Nature 591, 665–670 (2021).
Ortega, P., Merida-Cerro, J. A., Rondon, A. G., Gomez-Gonzalez, B. & Aguilera, A. DNA-RNA hybrids at DSBs interfere with repair by homologous recombination. eLife 10, e69881 (2021).
Zhao, H., Zhu, M., Limbo, O. & Russell, P. RNase H eliminates R-loops that disrupt DNA replication but is nonessential for efficient DSB repair. EMBO Rep. 19, e45335 (2018).
Sessa, G. et al. BRCA2 promotes DNA-RNA hybrid resolution by DDX5 helicase at DNA breaks to facilitate their repair. EMBO J. 40, e106018 (2021).
McBride, M. J. et al. The SS18-SSX fusion oncoprotein hijacks BAF complex targeting and function to drive synovial sarcoma. Cancer Cell 33, 1128–1141.e7 (2018).
Bauer, M. et al. The ALPK1/TIFA/NF-kappaB axis links a bacterial carcinogen to R-loop-induced replication stress. Nat. Commun. 11, 5117 (2020).
Nguyen, H. D. et al. Spliceosome mutations induce R loop-associated sensitivity to ATR inhibition in myelodysplastic syndromes. Cancer Res. 78, 5363–5374 (2018).
Herold, S. et al. Recruitment of BRCA1 limits MYCN-driven accumulation of stalled RNA polymerase. Nature 567, 545–549 (2019).
Schwab, R. A. et al. The Fanconi anemia pathway maintains genome stability by coordinating replication and transcription. Mol. Cell 60, 351–361 (2015).
Yeo, A. J. et al. R-loops in proliferating cells but not in the brain: implications for AOA2 and other autosomal recessive ataxias. PLoS One 9, e90219 (2014).
Lovejoy, C. A. et al. Loss of ATRX, genome instability, and an altered DNA damage response are hallmarks of the alternative lengthening of telomeres pathway. PLoS Genet. 8, e1002772 (2012).
Goldberg, A. D. et al. Distinct factors control histone variant H3.3 localization at specific genomic regions. Cell 140, 678–691 (2010).
Clynes, D. et al. Suppression of the alternative lengthening of telomere pathway by the chromatin remodelling factor ATRX. Nat. Commun. 6, 7538 (2015).
Wang, Y. et al. G-quadruplex DNA drives genomic instability and represents a targetable molecular abnormality in ATRX-deficient malignant glioma. Nat. Commun. 10, 943 (2019).
Zhang, J. M. & Zou, L. Alternative lengthening of telomeres: from molecular mechanisms to therapeutic outlooks. Cell Biosci. 10, 30 (2020).
Zhang, J. M., Genois, M. M., Ouyang, J., Lan, L. & Zou, L. Alternative lengthening of telomeres is a self-perpetuating process in ALT-associated PML bodies. Mol. Cell 81, 1027–1042.e4 (2021).
Vohhodina, J. et al. BRCA1 binds TERRA RNA and suppresses R-Loop-based telomeric DNA damage. Nat. Commun. 12, 3542 (2021).
Shiromoto, Y., Sakurai, M., Minakuchi, M., Ariyoshi, K. & Nishikura, K. ADAR1 RNA editing enzyme regulates R-loop formation and genome stability at telomeres in cancer cells. Nat. Commun. 12, 1654 (2021).
Ghisays, F. et al. RTEL1 influences the abundance and localization of TERRA RNA. Nat. Commun. 12, 3016 (2021).
Becherel, O. J. et al. A new model to study neurodegeneration in ataxia oculomotor apraxia type 2. Hum. Mol. Genet. 24, 5759–5774 (2015).
Katyal, S. et al. Aberrant topoisomerase-1 DNA lesions are pathogenic in neurodegenerative genome instability syndromes. Nat. Neurosci. 17, 813–821 (2014).
Grunseich, C. et al. Senataxin mutation reveals how R-loops promote transcription by Blocking DNA methylation at gene promoters. Mol. Cell 69, 426–437.e7 (2018).
Groh, M., Lufino, M. M., Wade-Martins, R. & Gromak, N. R-loops associated with triplet repeat expansions promote gene silencing in Friedreich ataxia and fragile X syndrome. PLoS Genet. 10, e1004318 (2014).
Haeusler, A. R. et al. C9orf72 nucleotide repeat structures initiate molecular cascades of disease. Nature 507, 195–200 (2014).
Wood, M. et al. TDP-43 dysfunction results in R-loop accumulation and DNA replication defects. J. Cell Sci. 133, jcs244129 (2020).
Walker, C. et al. C9orf72 expansion disrupts ATM-mediated chromosomal break repair. Nat. Neurosci. 20, 1225–1235 (2017).
Park, K. et al. Aicardi-Goutieres syndrome-associated gene SAMHD1 preserves genome integrity by preventing R-loop formation at transcription-replication conflict regions. PLoS Genet. 17, e1009523 (2021).
Mackenzie, K. J. et al. Ribonuclease H2 mutations induce a cGAS/STING-dependent innate immune response. EMBO J. 35, 831–844 (2016).
Coquel, F. et al. SAMHD1 acts at stalled replication forks to prevent interferon induction. Nature 557, 57–61 (2018).
Lee, S. C. et al. Modulation of splicing catalysis for therapeutic targeting of leukemia with mutations in genes encoding spliceosomal proteins. Nat. Med. 22, 672–678 (2016).
Cheruiyot, A. et al. Nonsense-mediated RNA decay is a unique vulnerability of cancer cells harboring SF3B1 or U2AF1 mutations. Cancer Res. 81, 4499–4513 (2021).
De Magis, A. et al. DNA damage and genome instability by G-quadruplex ligands are mediated by R loops in human cancer cells. Proc. Natl Acad. Sci. USA 116, 816–825 (2019).
Amato, R. et al. G-quadruplex stabilization fuels the ALT pathway in ALT-positive osteosarcoma cells. Genes 11, 304 (2020).
Safari, M. et al. R-loop-mediated ssDNA breaks accumulate following short-term exposure to the HDAC inhibitor romidepsin. Mol. Cancer Res. 19, 1361–1374 (2021).
Karanam, N. K., Ding, L., Aroumougame, A. & Story, M. D. Tumor treating fields cause replication stress and interfere with DNA replication fork maintenance: implications for cancer therapy. Transl. Res. 217, 33–46 (2020).
Tumini, E. et al. The antitumor drugs trabectedin and lurbinectedin induce transcription-dependent replication stress and genome instability. Mol. Cancer Res. 17, 773–782 (2019).
Petti, E. et al. SFPQ and NONO suppress RNA:DNA-hybrid-related telomere instability. Nat. Commun. 10, 1001 (2019).
Gomez-Gonzalez, B., Felipe-Abrio, I. & Aguilera, A. The S-phase checkpoint is required to respond to R-loops accumulated in THO mutants. Mol. Cell. Biol. 29, 5203–5213 (2009).
Gomez-Gonzalez, B. et al. Genome-wide function of THO/TREX in active genes prevents R-loop-dependent replication obstacles. EMBO J. 30, 3106–3119 (2011).
Nowotny, M. et al. Structure of human RNase H1 complexed with an RNA/DNA hybrid: insight into HIV reverse transcription. Mol. Cell 28, 264–276 (2007).
Rychlik, M. P. et al. Crystal structures of RNase H2 in complex with nucleic acid reveal the mechanism of RNA-DNA junction recognition and cleavage. Mol. Cell 40, 658–670 (2010).
Chang, E. Y. et al. RECQ-like helicases Sgs1 and BLM regulate R-loop-associated genome instability. J. Cell Biol. 216, 3991–4005 (2017).
Domingues-Silva, B., Silva, B. & Azzalin, C. M. Alternative functions for human FANCM at telomeres. Front. Mol. Biosci. 6, 84 (2019).
Mersaoui, S. Y. et al. Arginine methylation of the DDX5 helicase RGG/RG motif by PRMT5 regulates resolution of RNA:DNA hybrids. EMBO J. 38, e100986 (2019).
Garcia-Rubio, M. L. et al. The fanconi anemia pathway protects genome integrity from R-loops. PLoS Genet. 11, e1005674 (2015).
Lam, F. C. et al. BRD4 prevents the accumulation of R-loops and protects against transcription-replication collision events and DNA damage. Nat. Commun. 11, 4083 (2020).
We apologize to those authors whose work could not be cited due to space constrains. E.P. is supported by Cancer Research UK (C25526/A28275) and Medical Research Council (MR/S021310/1). L.L. is supported by the NIH (GM118833). L.Z. is the James & Patricia Poitras Endow Chair for Cancer Research and support by the NIH (CA263934).
The authors declare no competing interests.
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- Transcription–replication conflicts
Refers to collisions between transcription and DNA replication complexes and their consequences in both transcription and DNA replication.
- Class switch recombination
The process of switching the antibody type produced by mature B cells, during which the immunoglobulin gene is subject to transcription-dependent DNA damage followed by repair-mediated rearrangements.
- C-rich strand of telomere DNA
Telomere DNA consists of TTAGGG repeats on the G-rich strand and CCCTAA repeats on the C-rich strand.
- Replication stress
A plethora of DNA replication impediments that compromise the efficiency or fidelity of DNA synthesis and increase genomic instability.
- Control region of mtDNA
A non-coding region of the mitochondrial genome that controls RNA and DNA synthesis.
- Replication fork reversal
Backward movement of the replication fork during which the nascent newly synthesized strands dissociate from the template strands and anneal together to form a four-way junction.
(G4s). DNA secondary structures formed by guanine (G)-rich sequences through G–G base pairing.
Backward movement of transcribing RNA polymerase, which enables proofreading and regulation of transcript elongation.
- Integrator complex
A multisubunit protein complex with RNA endonuclease activity that controls the expression and processing of RNA polymerase II (Pol II) transcripts.
- RNA exosome
A multisubunit protein complex with 3′-5′exoribonuclease activity that degrades non-coding Pol II transcripts.
- Break-induced replication
(BIR). A process in which one-ended DNA breaks generated at replication forks are extended using homologous DNA as template.
- DNA end resection
A process in which exonucleases cleave one of the DNA strands at DNA double-stranded breaks to generate overhangs.
- Donor DNA
The DNA sequence used as template for DNA repair during homologous recombination.
- Translesion synthesis
A process in which a group of specialized DNA polymerases at or behind replication forks bypass DNA lesions to ensue replication.
- RNA editing
Post-transcriptional enzymatic process that changes RNA nucleotides, for example, by deaminating adenosine to inosine.
- Cerebellar ataxias
Progressive neurological disorders caused by damage to the cerebellum, characterized by inability to control balance, gait and muscle coordination.
- cGAS–STING pathway
Cellular signalling pathway that senses DNA in the cytoplasm as a sign of viral or bacterial infection and activates innate immunity responses.
- Nonsense-mediated mRNA decay
(NMD). A cellular surveillance mechanism that detects and degrades mRNAs harbouring premature termination codons.
- Tumour-treating electric fields
A non-invasive treatment, in which alternating electric fields are applied to tumour sites to inhibit cancer cell proliferation.
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Petermann, E., Lan, L. & Zou, L. Sources, resolution and physiological relevance of R-loops and RNA–DNA hybrids. Nat Rev Mol Cell Biol 23, 521–540 (2022). https://doi.org/10.1038/s41580-022-00474-x
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