Immunoprecipitation of RNA:DNA Hybrids from Budding Yeast

  • Aziz El HageEmail author
  • David Tollervey
Part of the Methods in Molecular Biology book series (MIMB, volume 1703)


During transcription, the nascent transcript behind an elongating RNA polymerase (RNAP) can invade the DNA duplex and hybridize with the complementary DNA template strand, generating a three-stranded “R-loop” structure, composed of an RNA:DNA duplex and an unpaired non-template DNA strand. R-loops can be strongly associated with actively transcribed loci by all RNAPs including the mitochondrial RNA polymerase (mtRNAP). In this chapter, we describe two protocols for the detection of RNA:DNA hybrids in living budding yeast cells, one that uses conventional chromatin immunoprecipitation (ChIP-qPCR) and one that uses DNA:RNA immunoprecipitation (DRIP-qPCR). Both protocols make use of the S9.6 antibody, which is believed to recognize the intermediate A/B helical RNA:DNA duplex conformation, with no sequence specificity.

Key words

Saccharomyces cerevisiae RNA:DNA hybrids R-loop Chromatin immunoprecipitation (ChIP) DNA:RNA immunoprecipitation (DRIP) S9.6 antibody 



We thank Kim Kotovic for initial help with the ChIP technique, members of Jean Beggs lab for giving us access to the bioruptor PICO, and Shaun Webb for help with bioinformatics analysis. We thank Andres Aguilera, Frederic Chedin, Martin Reijns, and Leonel Sanz for sharing protocols and/or reagents. We thank Frederic Chedin, Benoit Palancade, and Ralf Wellinger for critically reading the manuscript. We apologise to the colleagues whose work is not cited in this chapter due to space constraints. This work was supported by a Wellcome Trust Fellowship to DT (077248) and by core funding to the Wellcome Trust Centre for Cell Biology (092076).


  1. 1.
    Lesage P, Todeschini AL (2005) Happy together: the life and times of Ty retrotransposons and their hosts. Cytogenet Genome Res 110(1–4):70–90CrossRefPubMedGoogle Scholar
  2. 2.
    Lujan SA, Williams JS, Kunkel TA (2016) DNA polymerases divide the labor of genome replication. Trends Cell Biol 26(9):640–654CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Nudler E (2009) RNA polymerase active center: the molecular engine of transcription. Annu Rev Biochem 78:335–61. doi: Review. PMID:19489723
  4. 4.
    Aguilera A, Garcia-Muse T (2012) R loops: from transcription byproducts to threats to genome stability. Mol Cell 46(2):115–124CrossRefPubMedGoogle Scholar
  5. 5.
    Drolet M (2006) Growth inhibition mediated by excess negative supercoiling: the interplay between transcription elongation, R-loop formation and DNA topology. Mol Microbiol 59(3):723–730CrossRefPubMedGoogle Scholar
  6. 6.
    Groh M, Gromak N (2014) Out of balance: R-loops in human disease. PLoS Genet 10(9):e1004630CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Hamperl S, Cimprich KA (2014) The contribution of co-transcriptional RNA:DNA hybrid structures to DNA damage and genome instability. DNA Repair (Amst) 19:84–94CrossRefGoogle Scholar
  8. 8.
    Santos-Pereira JM, Aguilera A (2015) R loops: new modulators of genome dynamics and function. Nat Rev Genet 16(10):583–597CrossRefPubMedGoogle Scholar
  9. 9.
    Skourti-Stathaki K, Proudfoot NJ (2014) A double-edged sword: R loops as threats to genome integrity and powerful regulators of gene expression. Genes Dev 28(13):1384–1396CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Yu K et al (2003) R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells. Nat Immunol 4(5):442–451CrossRefPubMedGoogle Scholar
  11. 11.
    Pefanis E, Basu U (2015) RNA exosome regulates AID DNA Mutator activity in the B cell genome. Adv Immunol 127:257–308CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Drolet M et al (1995) Overexpression of RNase H partially complements the growth defect of an Escherichia coli delta topA mutant: R-loop formation is a major problem in the absence of DNA topoisomerase I. Proc Natl Acad Sci U S A 92(8):3526–3530CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Pommier Y et al (2016) Roles of eukaryotic topoisomerases in transcription, replication and genomic stability. Nat Rev Mol Cell Biol 17(11):703–721CrossRefPubMedGoogle Scholar
  14. 14.
    Fernandez X et al (2014) Chromatin regulates DNA torsional energy via topoisomerase II-mediated relaxation of positive supercoils. EMBO J 33(13):1492–1501PubMedPubMedCentralGoogle Scholar
  15. 15.
    El Hage A et al (2010) Loss of topoisomerase I leads to R-loop-mediated transcriptional blocks during ribosomal RNA synthesis. Genes Dev 24(14):1546–1558CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    French SL et al (2011) Distinguishing the roles of topoisomerases I and II in relief of transcription-induced torsional stress in yeast rRNA genes. Mol Cell Biol 31(3):482–494CrossRefPubMedGoogle Scholar
  17. 17.
    Cerritelli SM, Crouch RJ (2009) Ribonuclease H: the enzymes in eukaryotes. FEBS J 276(6):1494–1505CrossRefPubMedGoogle Scholar
  18. 18.
    Stuckey R et al (2015) Role for RNA:DNA hybrids in origin-independent replication priming in a eukaryotic system. Proc Natl Acad Sci U S A 112(18):5779–5784CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Amon JD, Koshland D (2016) RNase H enables efficient repair of R-loop induced DNA damage. elife 5.
  20. 20.
    Christman MF, Dietrich FS, Fink GR (1988) Mitotic recombination in the rDNA of S. cerevisiae is suppressed by the combined action of DNA topoisomerases I and II. Cell 55(3):413–425CrossRefPubMedGoogle Scholar
  21. 21.
    Salvi JS et al (2014) Roles for Pbp1 and caloric restriction in genome and lifespan maintenance via suppression of RNA-DNA hybrids. Dev Cell 30(2):177–191CrossRefPubMedGoogle Scholar
  22. 22.
    Pannunzio NR, Lieber MR (2016) Dissecting the roles of divergent and convergent transcription in chromosome instability. Cell Rep 14(5):1025–1031CrossRefPubMedGoogle Scholar
  23. 23.
    Yadav P, Owiti N, Kim N (2016) The role of topoisomerase I in suppressing genome instability associated with a highly transcribed guanine-rich sequence is not restricted to preventing RNA:DNA hybrid accumulation. Nucleic Acids Res 44(2):718–729CrossRefPubMedGoogle Scholar
  24. 24.
    Jenjaroenpun P et al (2017) R-loopDB: a database for R-loop forming sequences (RLFS) and R-loops. Nucleic Acids Res 45(D1):D119–D127CrossRefPubMedGoogle Scholar
  25. 25.
    Halasz L et al (2017) RNA-DNA hybrid (R-loop) immunoprecipitation mapping: an analytical workflow to evaluate inherent biases. Genome Res 27(6):1063–1073CrossRefPubMedGoogle Scholar
  26. 26.
    Boguslawski SJ et al (1986) Characterization of monoclonal antibody to DNA.RNA and its application to immunodetection of hybrids. J Immunol Methods 89(1):123–130CrossRefPubMedGoogle Scholar
  27. 27.
    El Hage A et al (2014) Genome-wide distribution of RNA-DNA hybrids identifies RNase H targets in tRNA genes, retrotransposons and mitochondria. PLoS Genet 10(10):e1004716CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Rigby RE et al (2014) RNA:DNA hybrids are a novel molecular pattern sensed by TLR9. EMBO J 33(6):542–558CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Koo CX et al (2015) RNA polymerase III regulates cytosolic RNA:DNA hybrids and intracellular microRNA expression. J Biol Chem 290(12):7463–7473CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Hu Z et al (2006) An antibody-based microarray assay for small RNA detection. Nucleic Acids Res 34(7):e52CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Chan YA et al (2014) Genome-wide profiling of yeast DNA:RNA hybrid prone sites with DRIP-chip. PLoS Genet 10(4):e1004288CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Legros P et al (2014) RNA processing factors Swd2.2 and Sen1 antagonize RNA Pol III-dependent transcription and the localization of condensin at Pol III genes. PLoS Genet 10(11):e1004794CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Saini N et al (2017) APOBEC3B cytidine deaminase targets the non-transcribed strand of tRNA genes in yeast. DNA Repair (Amst) 53:4–14CrossRefGoogle Scholar
  34. 34.
    Mischo HE et al (2011) Yeast Sen1 helicase protects the genome from transcription-associated instability. Mol Cell 41(1):21–32CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Alzu A et al (2012) Senataxin associates with replication forks to protect fork integrity across RNA-polymerase-II-transcribed genes. Cell 151(4):835–846CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Grzechnik P, Gdula MR, Proudfoot NJ (2015) Pcf11 orchestrates transcription termination pathways in yeast. Genes Dev 29(8):849–861CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Balk B et al (2013) Telomeric RNA-DNA hybrids affect telomere-length dynamics and senescence. Nat Struct Mol Biol 20(10):1199–1205CrossRefPubMedGoogle Scholar
  38. 38.
    Pfeiffer V et al (2013) The THO complex component Thp2 counteracts telomeric R-loops and telomere shortening. EMBO J 32(21):2861–2871CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Cloutier SC et al (2016) Regulated formation of lncRNA-DNA hybrids enables faster transcriptional induction and environmental adaptation. Mol Cell 61(3):393–404CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Keskin H, Meers C, Storici F (2016) Transcript RNA supports precise repair of its own DNA gene. RNA Biol 13(2):157–165CrossRefPubMedGoogle Scholar
  41. 41.
    Ohle C et al (2016) Transient RNA-DNA hybrids are required for efficient double-strand break repair. Cell 167(4):1001–1013.e7. CrossRefPubMedGoogle Scholar
  42. 42.
    Holmes JB et al (2015) Primer retention owing to the absence of RNase H1 is catastrophic for mitochondrial DNA replication. Proc Natl Acad Sci U S A 112(30):9334–9339CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Wahba L et al (2016) S1-DRIP-seq identifies high expression and polyA tracts as major contributors to R-loop formation. Genes Dev 30(11):1327–1338CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Sanz LA et al (2016) Prevalent, dynamic, and conserved R-loop structures associate with specific Epigenomic signatures in mammals. Mol Cell 63(1):167–178CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Chedin F (2016) Nascent connections: R-loops and chromatin patterning. Trends Genet 32(12):828–838CrossRefPubMedGoogle Scholar
  46. 46.
    Santos-Pereira JM et al (2013) The Npl3 hnRNP prevents R-loop-mediated transcription-replication conflicts and genome instability. Genes Dev 27(22):2445–2458CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Kim N, Jinks-Robertson S (2012) Transcription as a source of genome instability. Nat Rev Genet 13(3):204–214PubMedPubMedCentralGoogle Scholar
  48. 48.
    Wang IX et al (2016) RNA-DNA sequence differences in Saccharomyces cerevisiae. Genome Res 26(11):1544–1554CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Phillips DD et al (2013) The sub-nanomolar binding of DNA-RNA hybrids by the single-chain Fv fragment of antibody S9.6. J Mol Recognit 26(8):376–381CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Aparicio O et al (2005) Chromatin immunoprecipitation for determining the association of proteins with specific genomic sequences in vivo. Curr Protoc Mol Biol Chapter 21: p. Unit 21 3Google Scholar
  51. 51.
    Ginno PA et al (2012) R-loop formation is a distinctive characteristic of unmethylated human CpG island promoters. Mol Cell 45(6):814–825CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Zhao DY et al (2016) SMN and symmetric arginine dimethylation of RNA polymerase II C-terminal domain control termination. Nature 529(7584):48–53CrossRefPubMedGoogle Scholar
  53. 53.
    Brown TA, Tkachuk AN, Clayton DA (2008) Native R-loops persist throughout the mouse mitochondrial DNA genome. J Biol Chem 283(52):36743–36751CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.Wellcome Centre for Cell BiologyUniversity of EdinburghEdinburghUK

Personalised recommendations