R-loops are three-stranded structures generated by annealing of nascent transcripts to the template DNA strand, leaving the non-template DNA strand exposed as a single-stranded loop. Although R-loops play important roles in physiological processes such as regulation of gene expression, mitochondrial DNA replication, or immunoglobulin class switch recombination, dysregulation of the R-loop metabolism poses a threat to the stability of the genome. A previous study in yeast has shown that the homologous recombination machinery contributes to the formation of R-loops and associated chromosome instability. On the contrary, here, we demonstrate that depletion of the key homologous recombination factor, RAD51, as well as RAD51 inhibition by the B02 inhibitor did not prevent R-loop formation induced by the inhibition of spliceosome assembly in human cells. However, we noticed that treatment of cells with B02 resulted in RAD51-dependent accumulation of R-loops in an early G1 phase of the cell cycle accompanied by a decrease in the levels of chromatin-bound ORC2 protein, a component of the pre-replication complex, and an increase in DNA synthesis. Our results suggest that B02-induced R-loops might cause a premature origin firing.

Faculty of Science Charles University Prague 12800 Prague Czech Republic

Institute of Molecular Cancer Research University of Zurich 8057 Zurich Switzerland

Institute of Molecular Genetics of the Czech Academy of Sciences 14220 Prague Czech Republic

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Thomas M., White R.L., Davis R.W. Hybridization of RNA to double-stranded DNA: Formation of R-loops. Proc. Natl. Acad. Sci. USA. 1976;73:2294–2298. doi: 10.1073/pnas.73.7.2294. PubMed DOI PMC

Feretzaki M., Pospisilova M., Valador Fernandes R., Lunardi T., Krejci L., Lingner J. RAD51-dependent recruitment of TERRA lncRNA to telomeres through R-loops. Nature. 2020;587:303–308. doi: 10.1038/s41586-020-2815-6. PubMed DOI PMC

Sollier J., Cimprich K.A. Breaking bad: R-loops and genome integrity. Trends Cell Biol. 2015;25:514–522. doi: 10.1016/j.tcb.2015.05.003. PubMed DOI PMC

Garcia-Muse T., Aguilera A. R Loops: From Physiological to Pathological Roles. Cell. 2019;179:604–618. doi: 10.1016/j.cell.2019.08.055. PubMed DOI

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. 2012;45:814–825. doi: 10.1016/j.molcel.2012.01.017. PubMed DOI PMC

Chen L., Chen J.Y., Zhang X., Gu Y., Xiao R., Shao C., Tang P., Qian H., Luo D., Li H., et al. R-ChIP Using Inactive RNase H Reveals Dynamic Coupling of R-loops with Transcriptional Pausing at Gene Promoters. Mol. Cell. 2017;68:745–757. doi: 10.1016/j.molcel.2017.10.008. PubMed DOI PMC

Wahba L., Costantino L., Tan F.J., Zimmer A., Koshland D. S1-DRIP-seq identifies high expression and polyA tracts as major contributors to R-loop formation. Genes Dev. 2016;30:1327–1338. doi: 10.1101/gad.280834.116. PubMed DOI PMC

Ginno P.A., Lim Y.W., Lott P.L., Korf I., Chedin F. GC skew at the 5′ and 3′ ends of human genes links R-loop formation to epigenetic regulation and transcription termination. Genome Res. 2013;23:1590–1600. doi: 10.1101/gr.158436.113. PubMed DOI PMC

Boque-Sastre R., Soler M., Oliveira-Mateos C., Portela A., Moutinho C., Sayols S., Villanueva A., Esteller M., Guil S. Head-to-head antisense transcription and R-loop formation promotes transcriptional activation. Proc. Natl. Acad. Sci. USA. 2015;112:5785–5790. doi: 10.1073/pnas.1421197112. PubMed DOI PMC

Skourti-Stathaki K., Kamieniarz-Gdula K., Proudfoot N.J. R-loops induce repressive chromatin marks over mammalian gene terminators. Nature. 2014;516:436–439. doi: 10.1038/nature13787. PubMed DOI PMC

Hamperl S., Cimprich K.A. The contribution of co-transcriptional RNA:DNA hybrid structures to DNA damage and genome instability. DNA Repair Amst. 2014;19:84–94. doi: 10.1016/j.dnarep.2014.03.023. PubMed DOI PMC

Hamperl S., Cimprich K.A. Conflict Resolution in the Genome: How Transcription and Replication Make It Work. Cell. 2016;167:1455–1467. doi: 10.1016/j.cell.2016.09.053. PubMed DOI PMC

Helmrich A., Ballarino M., Nudler E., Tora L. Transcription-replication encounters, consequences and genomic instability. Nat. Struct. Mol. Biol. 2013;20:412–418. doi: 10.1038/nsmb.2543. PubMed DOI

Huertas P., Aguilera A. Cotranscriptionally formed DNA:RNA hybrids mediate transcription elongation impairment and transcription-associated recombination. Mol. Cell. 2003;12:711–721. doi: 10.1016/j.molcel.2003.08.010. PubMed DOI

Li X., Manley J.L. Inactivation of the SR protein splicing factor ASF/SF2 results in genomic instability. Cell. 2005;122:365–378. doi: 10.1016/j.cell.2005.06.008. PubMed DOI

Cerritelli S.M., Crouch R.J. Ribonuclease H: The enzymes in eukaryotes. FEBS J. 2009;276:1494–1505. doi: 10.1111/j.1742-4658.2009.06908.x. PubMed DOI PMC

Nowotny M., Gaidamakov S.A., Ghirlando R., Cerritelli S.M., Crouch R.J., Yang W. Structure of human RNase H1 complexed with an RNA/DNA hybrid: Insight into HIV reverse transcription. Mol. Cell. 2007;28:264–276. doi: 10.1016/j.molcel.2007.08.015. PubMed DOI

Groh M., Albulescu L.O., Cristini A., Gromak N. Senataxin: Genome Guardian at the Interface of Transcription and Neurodegeneration. J. Mol. Biol. 2017;429:3181–3195. doi: 10.1016/j.jmb.2016.10.021. PubMed DOI

Kasahara M., Clikeman J.A., Bates D.B., Kogoma T. RecA protein-dependent R-loop formation in vitro. Genes Dev. 2000;14:360–365. PubMed PMC

Zaitsev E.N., Kowalczykowski S.C. A novel pairing process promoted by Escherichia coli RecA protein: Inverse DNA and RNA strand exchange. Genes Dev. 2000;14:740–749. PubMed PMC

Huang F., Motlekar N.A., Burgwin C.M., Napper A.D., Diamond S.L., Mazin A.V. Identification of specific inhibitors of human RAD51 recombinase using high-throughput screening. ACS Chem. Biol. 2011;6:628–635. doi: 10.1021/cb100428c. PubMed DOI PMC

Huang F., Mazin A.V. A small molecule inhibitor of human RAD51 potentiates breast cancer cell killing by therapeutic agents in mouse xenografts. PLoS ONE. 2014;9:e100993. doi: 10.1371/journal.pone.0100993. PubMed DOI PMC

Huang F., Mazina O.M., Zentner I.J., Cocklin S., Mazin A.V. Inhibition of homologous recombination in human cells by targeting RAD51 recombinase. J. Med. Chem. 2012;55:3011–3020. doi: 10.1021/jm201173g. PubMed DOI

Urban V., Dobrovolna J., Huhn D., Fryzelkova J., Bartek J., Janscak P. RECQ5 helicase promotes resolution of conflicts between replication and transcription in human cells. J. Cell Biol. 2016;214:401–415. doi: 10.1083/jcb.201507099. PubMed DOI PMC

Boguslawski S.J., Smith D.E., Michalak M.A., Mickelson K.E., Yehle C.O., Patterson W.L., Carrico R.J. Characterization of monoclonal antibody to DNA.RNA and its application to immunodetection of hybrids. J. Immunol. Methods. 1986;89:123–130. doi: 10.1016/0022-1759(86)90040-2. PubMed DOI

Nguyen H.D., Yadav T., Giri S., Saez B., Graubert T.A., Zou L. Functions of Replication Protein A as a Sensor of R Loops and a Regulator of RNaseH1. Mol. Cell. 2017;65:832–847. doi: 10.1016/j.molcel.2017.01.029. PubMed DOI PMC

Teloni F., Michelena J., Lezaja A., Kilic S., Ambrosi C., Menon S., Dobrovolna J., Imhof R., Janscak P., Baubec T., et al. Efficient Pre-mRNA Cleavage Prevents Replication-Stress-Associated Genome Instability. Mol. Cell. 2019;73:670–683. doi: 10.1016/j.molcel.2018.11.036. PubMed DOI PMC

Wu H., Lima W.F., Crooke S.T. Investigating the structure of human RNase H1 by site-directed mutagenesis. J. Biol. Chem. 2001;276:23547–23553. doi: 10.1074/jbc.M009676200. PubMed DOI

Tsuzuki T., Fujii Y., Sakumi K., Tominaga Y., Nakao K., Sekiguchi M., Matsushiro A., Yoshimura Y., Morita T. Targeted disruption of the Rad51 gene leads to lethality in embryonic mice. Proc. Natl. Acad. Sci. USA. 1996;93:6236–6240. doi: 10.1073/pnas.93.13.6236. PubMed DOI PMC

Tazi J., Bakkour N., Soret J., Zekri L., Hazra B., Laine W., Baldeyrou B., Lansiaux A., Bailly C. Selective inhibition of topoisomerase I and various steps of spliceosome assembly by diospyrin derivatives. Mol. Pharmacol. 2005;67:1186–1194. doi: 10.1124/mol.104.007633. PubMed DOI

Tuduri S., Crabbe L., Conti C., Tourriere H., Holtgreve-Grez H., Jauch A., Pantesco V., De Vos J., Thomas A., Theillet C., et al. Topoisomerase I suppresses genomic instability by preventing interference between replication and transcription. Nat. Cell Biol. 2009;11:1315–1324. doi: 10.1038/ncb1984. PubMed DOI PMC

Aubry A., Pearson J.D., Huang K., Livne-Bar I., Ahmad M., Jagadeesan M., Khetan V., Ketela T., Brown K.R., Yu T., et al. Functional genomics identifies new synergistic therapies for retinoblastoma. Oncogene. 2020;39:5338–5357. doi: 10.1038/s41388-020-1372-7. PubMed DOI PMC

King H.O., Brend T., Payne H.L., Wright A., Ward T.A., Patel K., Egnuni T., Stead L.F., Patel A., Wurdak H., et al. RAD51 Is a Selective DNA Repair Target to Radiosensitize Glioma Stem Cells. Stem Cell Rep. 2017;8:125–139. doi: 10.1016/j.stemcr.2016.12.005. PubMed DOI PMC

Aguilera A., Garcia-Muse T. R loops: From transcription byproducts to threats to genome stability. Mol. Cell. 2012;46:115–124. doi: 10.1016/j.molcel.2012.04.009. PubMed DOI

Sanz L.A., Hartono S.R., Lim Y.W., Steyaert S., Rajpurkar A., Ginno P.A., Xu X., Chedin F. Prevalent, Dynamic, and Conserved R-Loop Structures Associate with Specific Epigenomic Signatures in Mammals. Mol. Cell. 2016;63:167–178. doi: 10.1016/j.molcel.2016.05.032. PubMed DOI PMC

Dumelie J.G., Jaffrey S.R. Defining the location of promoter-associated R-loops at near-nucleotide resolution using bisDRIP-seq. Elife. 2017;6 doi: 10.7554/eLife.28306. PubMed DOI PMC

Ratmeyer L., Vinayak R., Zhong Y.Y., Zon G., Wilson W.D. Sequence specific thermodynamic and structural properties for DNA.RNA duplexes. Biochemistry. 1994;33:5298–5304. doi: 10.1021/bi00183a037. PubMed DOI

El Hage A., Tollervey D. Immunoprecipitation of RNA:DNA Hybrids from Budding Yeast. Methods Mol. Biol. 2018;1703:109–129. doi: 10.1007/978-1-4939-7459-7_8. PubMed DOI

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. 2019;14:1661–1685. doi: 10.1038/s41596-019-0154-6. PubMed DOI PMC

Donovan S., Harwood J., Drury L.S., Diffley J.F. Cdc6p-dependent loading of Mcm proteins onto pre-replicative chromatin in budding yeast. Proc. Natl. Acad. Sci. USA. 1997;94:5611–5616. doi: 10.1073/pnas.94.11.5611. PubMed DOI PMC

Evrin C., Clarke P., Zech J., Lurz R., Sun J., Uhle S., Li H., Stillman B., Speck C. A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication. Proc. Natl. Acad. Sci. USA. 2009;106:20240–20245. doi: 10.1073/pnas.0911500106. PubMed DOI PMC

Remus D., Beuron F., Tolun G., Griffith J.D., Morris E.P., Diffley J.F. Concerted loading of Mcm2-7 double hexamers around DNA during DNA replication origin licensing. Cell. 2009;139:719–730. doi: 10.1016/j.cell.2009.10.015. PubMed DOI PMC

Siddiqui K., On K.F., Diffley J.F. Regulating DNA replication in eukarya. Cold Spring Harb. Perspect. Biol. 2013;5 doi: 10.1101/cshperspect.a012930. PubMed DOI PMC

Ticau S., Friedman L.J., Ivica N.A., Gelles J., Bell S.P. Single-molecule studies of origin licensing reveal mechanisms ensuring bidirectional helicase loading. Cell. 2015;161:513–525. doi: 10.1016/j.cell.2015.03.012. PubMed DOI PMC

Sollier J., Stork C.T., Garcia-Rubio M.L., Paulsen R.D., Aguilera A., Cimprich K.A. Transcription-coupled nucleotide excision repair factors promote R-loop-induced genome instability. Mol. Cell. 2014;56:777–785. doi: 10.1016/j.molcel.2014.10.020. PubMed DOI PMC

Marti T.M., Hefner E., Feeney L., Natale V., Cleaver J.E. H2AX phosphorylation within the G1 phase after UV irradiation depends on nucleotide excision repair and not DNA double-strand breaks. Proc. Natl. Acad. Sci. USA. 2006;103:9891–9896. doi: 10.1073/pnas.0603779103. PubMed DOI PMC

De Magis A., Manzo S.G., Russo M., Marinello J., Morigi R., Sordet O., Capranico G. DNA damage and genome instability by G-quadruplex ligands are mediated by R loops in human cancer cells. Proc. Natl. Acad. Sci. USA. 2019;116:816–825. doi: 10.1073/pnas.1810409116. PubMed DOI PMC

Carpenter A.E., Jones T.R., Lamprecht M.R., Clarke C., Kang I.H., Friman O., Guertin D.A., Chang J.H., Lindquist R.A., Moffat J., et al. CellProfiler: Image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 2006;7:R100. doi: 10.1186/gb-2006-7-10-r100. PubMed DOI PMC

Kamentsky L., Jones T.R., Fraser A., Bray M.A., Logan D.J., Madden K.L., Ljosa V., Rueden C., Eliceiri K.W., Carpenter A.E. Improved structure, function and compatibility for CellProfiler: Modular high-throughput image analysis software. Bioinformatics. 2011;27:1179–1180. doi: 10.1093/bioinformatics/btr095. PubMed DOI PMC

McQuin C., Goodman A., Chernyshev V., Kamentsky L., Cimini B.A., Karhohs K.W., Doan M., Ding L., Rafelski S.M., Thirstrup D., et al. CellProfiler 3.0: Next-generation image processing for biology. PLoS Biol. 2018;16:e2005970. doi: 10.1371/journal.pbio.2005970. PubMed DOI PMC

Masuda T., Tomita M., Ishihama Y. Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis. J. Proteome Res. 2008;7:731–740. doi: 10.1021/pr700658q. PubMed DOI

Rappsilber J., Mann M., Ishihama Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2007;2:1896–1906. doi: 10.1038/nprot.2007.261. PubMed DOI

Hebert A.S., Richards A.L., Bailey D.J., Ulbrich A., Coughlin E.E., Westphall M.S., Coon J.J. The one hour yeast proteome. Mol. Cell Proteom. 2014;13:339–347. doi: 10.1074/mcp.M113.034769. PubMed DOI PMC

Cox J., Hein M.Y., Luber C.A., Paron I., Nagaraj N., Mann M. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol. Cell Proteom. 2014;13:2513–2526. doi: 10.1074/mcp.M113.031591. PubMed DOI PMC

Tyanova S., Temu T., Sinitcyn P., Carlson A., Hein M.Y., Geiger T., Mann M., Cox J. The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat. Methods. 2016;13:731–740. doi: 10.1038/nmeth.3901. PubMed DOI

DDX17 helicase promotes resolution of R-loop-mediated transcription-replication conflicts in human cells