Abstract
Oncogene activation during tumorigenesis generates DNA replication stress, a known driver of genome rearrangements. In response to replication stress, certain loci, such as common fragile sites and telomeres, remain under-replicated during interphase and subsequently complete locus duplication in mitosis in a process known as ‘MiDAS’. Here, we demonstrate that RTEL1 (regulator of telomere elongation helicase 1) has a genome-wide role in MiDAS at loci prone to form G-quadruplex-associated R-loops, in a process that is dependent on its helicase function. We reveal that SLX4 is required for the timely recruitment of RTEL1 to the affected loci, which in turn facilitates recruitment of other proteins required for MiDAS, including RAD52 and POLD3. Our findings demonstrate that RTEL1 is required for MiDAS and suggest that RTEL1 maintains genome stability by resolving conflicts that can arise between the replication and transcription machineries.
Similar content being viewed by others
Data availability
The raw sequence data for each DRIP-seq sample are deposited at NCBI Bioproject (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA510030/). The names of the samples are YL_sample_7.fq.gz (siRNA-control), YL_sample_8.fq.gz (siRNA-control+RNase H), YL_sample_9.fq.gz (siRNA-RTEL1), YL_sample_10.fq.gz (siRNA-RTEL1+RNase H), YL_sample_11.fq.gz (siRNA-control+APH) and YL_sample_12.fq.gz (siRNA-control+APH+RNase H). The final processed data are deposited at the NCBI GEO database (accession no. GSE129907). The names of the samples are: sample_7.bed (siRNA-control), sample_8.bed (siRNA-control+RNase H), sample_9.bed (siRNA-RTEL1), sample_10.bed (siRNA-RTEL1+RNase H), sample_11.bed (siRNA-control+APH) and sample_12.bed (siRNA-control+APH+RNase H). The source data for graphs, western blots and DNA gels are available with the paper online. The source data for all of the images in figures are deposited at Figshare (https://doi.org/10.6084/m9.figshare.11833368).
Code availability
Custom-made Bash and Python scripts are available upon request from the corresponding authors.
References
Baddeley, D. et al. Measurement of replication structures at the nanometer scale using super-resolution light microscopy. Nucleic Acids Res 38, e8 (2010).
Arlt, M. F., Durkin, S. G., Ragland, R. L. & Glover, T. W. Common fragile sites as targets for chromosome rearrangements. DNA Repair 5, 1126–1135 (2006).
Burrell, R. A. et al. Replication stress links structural and numerical cancer chromosomal instability. Nature 494, 492–496 (2013).
Burrow, A. A., Williams, L. E., Pierce, L. C. & Wang, Y. H. Over half of breakpoints in gene pairs involved in cancer-specific recurrent translocations are mapped to human chromosomal fragile sites. BMC Genomics 10, 59 (2009).
Halazonetis, T. D., Gorgoulis, V. G. & Bartek, J. An oncogene-induced DNA damage model for cancer development. Science 319, 1352–1355 (2008).
Le Tallec, B. et al. Common fragile site profiling in epithelial and erythroid cells reveals that most recurrent cancer deletions lie in fragile sites hosting large genes. Cell Rep. 4, 420–428 (2013).
Dilley, R. L. et al. Break-induced telomere synthesis underlies alternative telomere maintenance. Nature 539, 54–58 (2016).
Min, J., Wright, W. E. & Shay, J. W. Alternative lengthening of telomeres mediated by mitotic DNA synthesis engages break-induced replication processes. Mol. Cell. Biol. 37, e00226-17 (2017).
Minocherhomji, S. et al. Replication stress activates DNA repair synthesis in mitosis. Nature 528, 286–290 (2015).
Özer, Ö., Bhowmick, R., Liu, Y. & Hickson, I. D. Human cancer cells utilize mitotic DNA synthesis to resist replication stress at telomeres regardless of their telomere maintenance mechanism. Oncotarget 9, 15836–15846 (2018).
Bhowmick, R., Minocherhomji, S. & Hickson, I. D. RAD52 facilitates mitotic DNA synthesis following replication stress. Mol. Cell 64, 1117–1126 (2016).
Letessier, A. et al. Cell-type-specific replication initiation programs set fragility of the FRA3B fragile site. Nature 470, 120–123 (2011).
Wilson, T. E. et al. Large transcription units unify copy number variants and common fragile sites arising under replication stress. Genome Res. 25, 189–200 (2015).
Helmrich, A., Ballarino, M. & Tora, L. Collisions between replication and transcription complexes cause common fragile site instability at the longest human genes. Mol. Cell 44, 966–977 (2011).
Santos-Pereira, J. M. & Aguilera, A. R loops: new modulators of genome dynamics and function. Nat. Rev. Genet. 16, 583–597 (2015).
Stirling, P. C. & Hieter, P. Canonical DNA repair pathways influence R-loop-driven genome instability. J. Mol. Biol. 429, 3132–3138 (2017).
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).
Fungtammasan, A., Walsh, E., Chiaromonte, F., Eckert, K. A. & Makova, K. D. A genome-wide analysis of common fragile sites: what features determine chromosomal instability in the human genome? Genome Res. 22, 993–1005 (2012).
Kuznetsov, V. A., Bondarenko, V., Wongsurawat, T., Yenamandra, S. P. & Jenjaroenpun, P. Toward predictive R-loop computational biology: genome-scale prediction of R-loops reveals their association with complex promoter structures, G-quadruplexes and transcriptionally active enhancers. Nucleic Acids Res. 46, 7566–7585 (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).
Pedersen, R. T., Kruse, T., Nilsson, J., Oestergaard, V. H. & Lisby, M. TopBP1 is required at mitosis to reduce transmission of DNA damage to G1 daughter cells. J. Cell Biol. 210, 565–582 (2015).
Vannier, J. B., Pavicic-Kaltenbrunner, V., Petalcorin, M. I., Ding, H. & Boulton, S. J. RTEL1 dismantles T loops and counteracts telomeric G4-DNA to maintain telomere integrity. Cell 149, 795–806 (2012).
Sotiriou, S. K. et al. Mammalian RAD52 functions in break-induced replication repair of collapsed DNA replication forks. Mol. Cell 64, 1127–1134 (2016).
Chan, Y. W., Fugger, K. & West, S. C. Unresolved recombination intermediates lead to ultra-fine anaphase bridges, chromosome breaks and aberrations. Nat. Cell Biol. 20, 92–103 (2018).
Liu, Y., Nielsen, C. F., Yao, Q. & Hickson, I. D. The origins and processing of ultra fine anaphase DNA bridges. Curr. Opin. Genet. Dev. 26, 1–5 (2014).
Nielsen, C. F. et al. PICH promotes sister chromatid disjunction and co-operates with topoisomerase II in mitosis. Nat. Commun. 6, 8962 (2015).
Lukas, C. et al. 53BP1 nuclear bodies form around DNA lesions generated by mitotic transmission of chromosomes under replication stress. Nat. Cell Biol. 13, 243–253 (2011).
Arora, R. et al. RNaseH1 regulates TERRA-telomeric DNA hybrids and telomere maintenance in ALT tumour cells. Nat. Commun. 5, 5220 (2014).
Hall, A. C., Ostrowski, L. A., Pietrobon, V. & Mekhail, K. Repetitive DNA loci and their modulation by the non-canonical nucleic acid structures R-loops and G-quadruplexes. Nucleus 8, 162–181 (2017).
Madireddy, A. et al. FANCD2 facilitates replication through common fragile sites. Mol. Cell 64, 388–404 (2016).
Barber, L. J. et al. RTEL1 maintains genomic stability by suppressing homologous recombination. Cell 135, 261–271 (2008).
Boguslawski, S. J. et al. Characterization of monoclonal-antibody to DNA · RNA and its application to immunodetection of hybrids. J. Immunol. Methods 89, 123–130 (1986).
Chan, K. L., Palmai-Pallag, T., Ying, S. M. & Hickson, I. D. Replication stress induces sister-chromatid bridging at fragile site loci in mitosis. Nat. Cell Biol. 11, 753–760 (2009).
Fan, Q., Zhang, F., Barrett, B., Ren, K. Q. & Andreassen, P. R. A role for monoubiquitinated FANCD2 at telomeres in ALT cells. Nucleic Acids Res. 37, 1740–1754 (2009).
Howlett, N. G., Taniguchi, T., Durkin, S. G., D’Andrea, A. D. & Glover, T. W. The Fanconi anemia pathway is required for the DNA replication stress response and for the regulation of common fragile site stability. Hum. Mol. Genet. 14, 693–701 (2005).
Sordet, O. et al. Ataxia telangiectasia mutated activation by transcription- and topoisomerase I-induced DNA double-strand breaks. EMBO Rep. 10, 887–893 (2009).
Tresini, M. et al. The core spliceosome as target and effector of non-canonical ATM signalling. Nature 523, 53–58 (2015).
Okamoto, Y. et al. Replication stress induces accumulation of FANCD2 at central region of large fragile genes. Nucleic Acids Res. 46, 2932–2944 (2018).
Glover, T. W., Berger, C., Coyle, J. & Echo, B. DNA polymerase-α inhibition by aphidicolin induces gaps and breaks at common fragile sites in human chromosomes. Hum. Genet. 67, 136–142 (1984).
Kumar, R. et al. HumCFS: a database of fragile sites in human chromosomes. BMC Genomics 19, 985 (2019).
Mrasek, K. et al. Global screening and extended nomenclature for 230 aphidicolin-inducible fragile sites, including 61 yet unreported ones. Int. J. Oncol. 36, 929–940 (2010).
Forbes, S. A. et al. COSMIC: somatic cancer genetics at high-resolution. Nucleic Acids Res. 45, D777–D783 (2017).
Kikin, O., D’Antonio, L. & Bagga, P. S. QGRS Mapper: a web-based server for predicting G-quadruplexes in nucleotide sequences. Nucleic Acids Res. 34, W676–W682 (2006).
Hansel-Hertsch, R., Spiegel, J., Marsico, G., Tannahill, D. & Balasubramanian, S. Genome-wide mapping of endogenous G-quadruplex DNA structures by chromatin immunoprecipitation and high-throughput sequencing. Nat. Protoc. 13, 551–564 (2018).
Zimmer, J. et al. Targeting BRCA1 and BRCA2 deficiencies with G-quadruplex-interacting compounds. Mol. Cell 61, 449–460 (2016).
Gavathiotis, E., Heald, R. A., Stevens, M. F. & Searle, M. S. Drug recognition and stabilisation of the parallel-stranded DNA quadruplex d(TTAGGGT)4 containing the human telomeric repeat. J. Mol. Biol. 334, 25–36 (2003).
Gowan, S. M., Heald, R., Stevens, M. F. & Kelland, L. R. Potent inhibition of telomerase by small-molecule pentacyclic acridines capable of interacting with G-quadruplexes. Mol. Pharmacol. 60, 981–988 (2001).
Heald, R. A. et al. Antitumor polycyclic acridines. 8.1 Synthesis and telomerase-inhibitory activity of methylated pentacyclic acridinium salts. J. Med. Chem. 45, 590–597 (2002).
Wu, X., Sandhu, S., Nabi, Z. & Ding, H. Generation of a mouse model for studying the role of upregulated RTEL1 activity in tumorigenesis. Transgenic Res. 21, 1109–1115 (2012).
Yamamoto, K. N. et al. Involvement of SLX4 in interstrand cross-link repair is regulated by the Fanconi anemia pathway. Proc. Natl Acad. Sci. USA 108, 6492–6496 (2011).
Wilson, J. S. et al. Localization-dependent and -independent roles of SLX4 in regulating telomeres. Cell Rep. 4, 853–860 (2013).
Parajuli, S. et al. Human ribonuclease H1 resolves R-loops and thereby enables progression of the DNA replication fork. J. Biol. Chem. 292, 15216–15224 (2017).
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).
Yuce, O. & West, S. C. Senataxin, defective in the neurodegenerative disorder ataxia with oculomotor apraxia 2, lies at the interface of transcription and the DNA damage response. Mol. Cell. Biol. 33, 406–417 (2013).
Zhao, H. C., 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).
Cohen, S. et al. Senataxin resolves RNA:DNA hybrids forming at DNA double-strand breaks to prevent translocations. Nat. Commun. 9, 533 (2018).
Aguilera, A. & Garcia-Muse, T. R Loops: from transcription byproducts to threats to genome stability. Mol. Cell 46, 115–124 (2012).
Hamperl, S. & Cimprich, K. A. The contribution of co-transcriptional RNA:DNA hybrid structures to DNA damage and genome instability. DNA Repair 19, 84–94 (2014).
Harrigan, J. A. et al. Replication stress induces 53BP1-containing OPT domains in G1 cells. J. Cell Biol. 193, 97–108 (2011).
Lemmens, B. et al. DNA replication determines timing of mitosis by restricting CDK1 and PLK1 activation. Mol. Cell 71, 117–128 (2018).
Macheret, M. & Halazonetis, T. D. Intragenic origins due to short G1 phases underlie oncogene-induced DNA replication stress. Nature 555, 112–116 (2018).
Munoz, I. M. et al. Coordination of structure-specific nucleases by human SLX4/BTBD12 is required for DNA repair. Mol. Cell 35, 116–127 (2009).
Takedachi, A. SLX4 interacts with RTEL1 to prevent transcription-mediated DNA replication perturbations Nat. Struct. Mol. Biol. https://doi.org/10.1038/s41594-020-0419-3 (2020).
Ballew, B. J. et al. Germline mutations of regulator of telomere elongation helicase 1, RTEL1, in dyskeratosis congenita. Hum. Genet. 132, 473–480 (2013).
Ballew, B. J. et al. A recessive founder mutation in regulator of telomere elongation helicase 1, RTEL1, underlies severe immunodeficiency and features of Hoyeraal Hreidarsson syndrome. PLoS Genet. 9, e1003695 (2013).
Deng, Z. et al. Inherited mutations in the helicase RTEL1 cause telomere dysfunction and Hoyeraal–Hreidarsson syndrome. Proc. Natl Acad. Sci. USA 110, E3408–E3416 (2013).
Egan, K. M. et al. Cancer susceptibility variants and the risk of adult glioma in a US case–control study. J. Neurooncol. 104, 535–542 (2011).
Liu, Y. et al. Polymorphisms of LIG4, BTBD2, HMGA2 and RTEL1 genes involved in the double-strand break repair pathway predict glioblastoma survival. J. Clin. Oncol. 28, 2467–2474 (2010).
Rajaraman, P. et al. Genome-wide association study of glioma and meta-analysis. Hum. Genet. 131, 1877–1888 (2012).
Shete, S. et al. Genome-wide association study identifies five susceptibility loci for glioma. Nat. Genet. 41, 899–904 (2009).
Wrensch, M. et al. Variants in the CDKN2B and RTEL1 regions are associated with high-grade glioma susceptibility. Nat. Genet. 41, 905–908 (2009).
Bai, C. et al. Overexpression of M68/DcR3 in human gastrointestinal tract tumors independent of gene amplification and its location in a four-gene cluster. Proc. Natl Acad. Sci. USA 97, 1230–1235 (2000).
Muleris, M., Almeida, A., Gerbaultseureau, M., Malfoy, B. & Dutrillaux, B. Identification of amplified DNA sequences in breast cancer and their organization within homogeneously staining regions. Genes Chromosomes Cancer 14, 155–163 (1995).
Pitti, R. M. et al. Genomic amplification of a decoy receptor for Fas ligand in lung and colon cancer. Nature 396, 699–703 (1998).
Garribba, L. et al. Inducing and detecting mitotic DNA synthesis at difficult-to-replicate oci. Methods Enzymol. 601, 45–58 (2018).
Bjerregaard, V. A., Garribba, L., McMurray, C. T., Hickson, I. D. & Liu, Y. Folate deficiency drives mitotic missegregation of the human FRAXA locus. Proc. Natl Acad. Sci. USA 115, 13003–13008 (2018).
Bizard, A. H., Nielsen, C. F. & Hickson, I. D. Detection of ultrafine anaphase bridges. Methods Mol. Biol. 1672, 495–508 (2018).
Skourti-Stathaki, K., Kamieniarz-Gdula, K. & Proudfoot, N. J. R-loops induce repressive chromatin marks over mammalian gene terminators. Nature 516, 436–439 (2014).
Biffi, G., Tannahill, D., McCafferty, J. & Balasubramanian, S. Quantitative visualization of DNA G-quadruplex structures in human cells. Nat. Chem. 5, 182–186 (2013).
Schwab, R. A. et al. The Fanconi anemia pathway maintains genome stability by coordinating replication and transcription. Mol. Cell 60, 351–361 (2015).
Acknowledgements
We thank members of the Hickson and Liu groups for helpful discussions, and J.P. Diaz and E. Hoffmann for critical reading of the manuscript. We also thank W. Qiao and V. Lee (BGI Hong Kong) for processing the DRIP-seq samples. This work was supported by grants from the Chinese Scholarship Council (PhD fellowship 201406170048; W.W.), the Danish Medical Research Council (Postdoc fellowship DFF-4004-00155B; R.B.), the European Commission (FP7 Marie Curie Fellowship; Ö.Ö.), US NIH grants (GM56888 and U54 OD020355; J.H.P.), US NCI MSK Cancer Center core grant P30 CA008748 (J.H.P.), The Nordea Foundation of Denmark (I.D.H.), The Danish National Research Foundation (DNRF115; I.D.H. and Y.L.) and the European Commission (H2020/ Marie Skłodowska-Curie, 859853; I.D.H. and Y.L.).
Author information
Authors and Affiliations
Contributions
W.W., R.B., Ö.Ö., F.G., R.S.T., E.S., P.H.R. and L.R. carried out experiments. I.V. performed analysis of bioinformatic data. W.W., R.B., J.H.P., I.D.H. and Y.L. designed experiments and interpreted results. I.D.H. and Y.L. wrote the manuscript and all authors edited it.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Peer review information Beth Moorefield was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data
Extended Data Fig. 1 RTEL1 depletion by 3’UTR siRNAs causes R-loop accumulation in interphase and reduced MiDAS in prometaphase cells under replication stress conditions.
a, Experimental workflow for the analysis of MiDAS in prometaphase cells in U2OS cells following RTEL1 depletion and APH treatment. b & c, Representative images of Immunoblotting analysis (b) and quantification (c) of RTEL1 expression following transfection with a control and two 3’UTR targeting siRNA. GAPDH was used as a loading control. The quantification of relative protein levels was performed using Fiji/ImageJ software. Error bars indicate mean ± s.d. (N = 3). **P < 0.01, based on two-tailed Student’s t-test. d & e, Representative IF images of three independent experiments (d) and quantification (e) of interphase cells treated with control siRNA or RTEL1 3’UTR siRNA for 48 hours and stained with S9.6 antibody (red) and FANCD2 antibody (green). A zoomed image for the area indicated by a white box is shown on the right. Error bars represent median ± s.e.m. (N = 3). ****p < 0.0001, based on two-tailed non-parametric Mann Whitney test. f & g, Representative IF images (f) and quantification (g) of MiDAS foci (labeled with EdU; red) in prometaphase cells treated as shown in panel a. DNA was stained with DAPI (blue). Error bars represent median ± s.e.m. (N = 3). ****p < 0.0001, based on two-tailed non-parametric Mann Whitney test. Scale bar is 10 µm. Data for graphs in panels c,e,g are available as Source data. Uncropped image for panel b is available as Source data.
Extended Data Fig. 2 RTEL1 is essential for MiDAS in telomerase positive cells under replication stress conditions.
a, Experimental workflow for the analysis of MiDAS in prometaphase HeLa or HCT116 cells following RTEL1 depletion and APH treatment. b & c, Representative images (b) and quantification (c) of RTEL1 expression following siRNA transfection with a control and RTEL1 siRNAs. Tubulin was used as a loading control. The quantification of relative protein levels was performed using Fiji/ImageJ software. Error bars indicate mean ± s.d. (N = 3). ****P < 0.0001, based on two-tailed Student’s t-test d & e, Representative IF images (d) and quantification (e) of MiDAS (indicated with EdU; red) in prometaphase cells. DNA was stained with DAPI (blue). Error bars represent median ± s.e.m. (N = 3). ****p < 0.0001, based on two-tailed non-parametric Mann Whitney test. Scale bar is 10 µm. Data for graphs in panels c,e are available as Source data. Uncropped image for panel b is available as Source data.
Extended Data Fig. 3 Following RTEL1 depletion, R-loops are accumulated in telomerase positive cells.
a, Experimental workflow for the analysis of R-loops in interphase HeLa or HCT116 cells following RTEL1 depletion. b & c, Representative IF images (b) and quantification (c) of interphase HeLa cells stained with S9.6 antibody specific for R-loops (red), or a nucleolin antibody (green). DNA was stained with DAPI (blue). Error bars represent median ± s.e.m. (N = 3). ****p < 0.0001, based on two-tailed non-parametric Mann Whitney test. d & e, Representative IF images (d) and quantification (e) of interphase HCT116 cells stained with S9.6 antibody specific for R-loops (red), or a nucleolin antibody (green). DNA was stained with DAPI (blue). The S9.6 signal intensity per nucleus was calculated by Fiji/ImageJ software and determined by subtracting the S9.6 staining with that from nucleoli (defined by nucleolin; green) in each nucleus. In each case, a specificity control for S9.6 staining is shown, in which fixed cells were treated with RNase H to eliminate R-loops. Error bars represent median ± s.e.m. (N = 3). ****p < 0.0001, based on two-tailed non-parametric Mann Whitney test. Scale bar is 10 µm. Data for graphs in panels c,e are available as Source data.
Extended Data Fig. 4 R-loops accumulate at specific regions of the genome following RTEL1 depletion.
a, Experimental workflow of cell synchronization and siRNA treatment for analyzing G1 phase U2OS cells. b, Representative IF images of G1 cells transfected with control siRNA or RTEL1 siRNA and stained with S9.6 antibody specific for R-loops (green) or a 53BP1 antibody (red). DNA was stained with DAPI (blue). Scale bar is 10 µm. c, Quantification of co-localization between 53BP1 bodies and R-loops in G1 cells from panel b. Error bars represent median ± s.e.m. (N = 3). ****p < 0.0001, based on two-tailed non-parametric Mann Whitney test. d, Experimental workflow for the quantitative analysis of R-loops enriched at different loci of the genome following RTEL1 depletion by DNA-RNA immunoprecipitation (DRIP) qPCR. e, Quantification of R-loops by DRIP qPCR at different genomic loci. The qPCR value was normalized against a region of the ZNF554 gene for each sample. Error bars indicate mean ± s.d. *p < 0.05; **p < 0.01, based on two-tailed Student’s t-test. (N for each locus: NRG3 = 8, FRA3B = 7, FRA16D = 4, FRA7B = 7, FRA10C = 3, 18 S = 5, 28 S = 3). Data for graphs in panels c,e are available as Source data.
Extended Data Fig. 5 RTEL1 depletion induces chromosome fragility at common fragile sites.
a, Experimental workflow for analyzing chromosome fragility in U2OS cells. b, Representative images of FISH signal on chromosomes from cells treated with either an siRNA control (siCon), an siRNA targeting RTEL1 (siRTEL1), or 0.4 μM APH. n, the number of loci examined. Loci of FRA16D (green) or FRA3B (red) were detected by FISH probes. DNA was stained with DAPI. c, Quantification of chromosome fragility at FRA16D or FRA3B. Error bars indicate mean ± s.d. (N = 3). *p < 0.05; **p < 0.01, based on two-tailed Student’s t-test. Scale bar is 2 µm. Data for graphs in panel c is available as Source data.
Extended Data Fig. 6 The analysis of un-bound protein and cell cycle profiles at different stages of interphase or mitosis following RTEL1 depletion and APH (0.4 µM) treatment.
The un-bound proteins was assessed by western blot analysis (a,c,e). GAPDH or Tubulin was used as loading controls. Histone H3 was used as a loading control for the chromatin-bound fraction. The cell cycle profiles (b,d,f) were output from Flowjo software and representative for two independent experiments. Uncropped images for panel a,c,e are available as Source data.
Extended Data Fig. 7 SLX4 depletion reduces the frequency of MiDAS in a U2OS cell line with inducible GFP-SLX4.
a, Experimental workflow of analyzing SLX4 in interphase cells or MiDAS in prometaphase cells by IF following SLX4 depletion. b, Representative images of a U2OS cell line with expression of GFP-SLX4 following the treatment with doxycycline (+Dox) to induce expression. GFP-SLX4 (green) or SLX4 (red) was detected by direct fluorescence (green; for GFP) or using antibodies against SLX4 respectively. c & d, Representative images of MiDAS (labeled with EdU; red) (c) and quantification (d) in prometaphase U2OS cells following SLX4 depletion by a 3’UTR siRNA or with inducible GFP-SLX4 expression. DNA was stained with DAPI (blue). Error bars represent median ± s.e.m. (N = 3). ****p < 0.0001, based on two-tailed non-parametric Mann Whitney test. Scale bar is 10 µm. Data for graphs in panel d is available as Source data.
Extended Data Fig. 8 SETX does not play a role in MiDAS.
a, Experimental workflow for analyzing prometaphase U2OS cells by IF following SETX depletion and APH treatment. b, Representative western blotting analysis of SETX following siRNA treatment. Actin was used as a loading control. c, d, Representative images of MiDAS (labeled with EdU; red) (c) and quantification (d) in prometaphase U2OS cells following SETX depletion. DNA was stained with DAPI (blue). Error bars represent median ± s.e.m. (N = 3). ns: p > 0.05, based on two-tailed non-parametric Mann Whitney test. Data for graphs in panel d is available as Source data. Uncropped image for panel b is available as Source data.
Extended Data Fig. 9 RNase H1 does not play a role in MiDAS.
a, Experimental workflow of analyzing interphase or prometaphase U2OS cells by IF following APH treatment and RNase H1 depletion alone or combined with RTEL1 depletion. b & c, Representative IF images (b) and quantification (c) of interphase cells stained with S9.6 antibody (red) and FANCD2 antibody (green). A zoomed image for the area indicated by a white box is shown on the right. Error bars represent median ± s.e.m. (N = 3). ****p < 0.0001, based on two-tailed non-parametric Mann Whitney test. d, Western blotting analysis of RNase H1 or RTEL1 following siRNA treatment. Actin was used as a loading control. e & f, Representative images of MiDAS (labeled with EdU; red) (e) and quantification (f) in prometaphase cells following RNaseH1 depletion alone or combined with RTEL1 depletion. Scale bar, 10 μm.DNA was stained with DAPI (blue). Error bars represent median ± s.e.m. (N = 3). ****p < 0.0001, based on two-tailed non-parametric Mann Whitney test. Data for graphs in panels c,f are available as Source data. Uncropped image for panel d is available as Source data.
Supplementary information
Supplementary Information
A cover page, reagents table, note, Figs. 1–3 and legends.
Supplementary Table 1
An Excel file of the peaks identified in DRIP-seq sample 7 (siRNA-control) and sample 9 (siRNA-RTEL1), excluding the peaks not affected by RNase H treatment. A P value was assigned to each peak, to assess whether it was differentially enriched in sample 9 against sample 7. In addition, these peaks are annotated with their genomic location and other features, including CFSs, G4 motifs, transcribed genes, COSMIC cancer mutations, COSMIC cancer gene expression and promoters.
Supplementary Table 2
An Excel file of the peaks identified in DRIP-seq sample 7 (siRNA-control) and sample 11 (siRNA-control+APH), excluding the peaks not affected by RNaseH treatment. A P value was assigned to each peak to assess whether it was differentially enriched in sample 11 against sample 7. In addition, these peaks have been annotated with their genomic location and other features, including CFSs, G4 motifs, transcribed genes, COSMIC cancer mutations, COSMIC cancer gene expression and promoters.
Supplementary Table 3
An Excel file of all of the CFSs annotated by three published databases in 2010, 2012 and 2019, respectively (see Supplementary Note).
Supplementary Data
Source data for unprocessed Western Blots and gels.
Source data
Source Data Fig. 1
Statistical source data
Source Data Fig. 2
Statistical source data
Source Data Fig. 3
Statistical source data
Source Data Fig. 4
Statistical source data
Source Data Fig. 5
Statistical source data
Source Data Fig. 6
Statistical source data
Source Data Fig. 7
Statistical source data
Source Data Fig. 8
Statistical source data
Source Data Extended Data Fig. 1
Statistical source data
Source Data Extended Data Fig. 2
Statistical source data
Source Data Extended Data Fig. 3
Statistical source data
Source Data Extended Data Fig. 4
Statistical source data
Source Data Extended Data Fig. 5
Statistical source data
Source Data Extended Data Fig. 7
Statistical source data
Source Data Extended Data Fig. 8
Statistical source data
Source Data Extended Data Fig. 9
Statistical source data
Rights and permissions
About this article
Cite this article
Wu, W., Bhowmick, R., Vogel, I. et al. RTEL1 suppresses G-quadruplex-associated R-loops at difficult-to-replicate loci in the human genome. Nat Struct Mol Biol 27, 424–437 (2020). https://doi.org/10.1038/s41594-020-0408-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41594-020-0408-6
- Springer Nature America, Inc.
This article is cited by
-
Genome-wide identification of replication fork stalling/pausing sites and the interplay between RNA Pol II transcription and DNA replication progression
Genome Biology (2024)
-
Telomouse—a mouse model with human-length telomeres generated by a single amino acid change in RTEL1
Nature Communications (2023)
-
Mitotic DNA synthesis in response to replication stress requires the sequential action of DNA polymerases zeta and delta in human cells
Nature Communications (2023)
-
Genetics of human telomere biology disorders
Nature Reviews Genetics (2023)
-
Excessive reactive oxygen species induce transcription-dependent replication stress
Nature Communications (2023)