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R-Loop Immunoprecipitation: A Method to Detect R-Loop Interacting Factors

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R-Loops

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2528))

Abstract

R-loops are non-B-DNA structures consisting of an RNA/DNA hybrid and a displaced single-stranded DNA. They arise during transcription and play important biological roles. However, perturbation of R-loop levels represents a source of DNA damage and genome instability resulting in human diseases, including cancer and neurodegeneration. In this chapter, we describe a protocol which allows detection of R-loop interactors using affinity purification with S9.6 antibody, specific for RNA/DNA hybrids, followed by Western blotting or mass spectrometry. Multiple specificity controls including addition of synthetic competitors and RNase H treatment are described to verify the specificity of identified R-loop-binding factors. The identification of new R-loop interacting factors and the characterization of their involvement in R-loop biology provides a powerful resource to study the role of these important structures in health and disease.

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Acknowledgments

We thank Maria Sole Giordano for testing this protocol and Prof. R.Harris for providing APOBEC3B antibody. We thank Holly Roach for editing this paper. This work was supported by the Royal Society University Research fellowship (BVD07340), Royal Society Enhancement Award (RGF\EA\180023), EPA Research Fund (Sir William Dunn School of Pathology, University of Oxford), Ataxia UK (ZGROMAK) and Rosetrees Fund (A2196) to NG. CB is supported by the Royal Society Enhancement Award (RGF\EA\180023) to NG and the Sir William Dunn School of Pathology, University of Oxford.

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Authors and Affiliations

  1. Sir William Dunn School of Pathology, University of Oxford, Oxford, UK

    Chiara Beghè & Natalia Gromak

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  1. Chiara Beghè
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  2. Natalia Gromak
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Correspondence to Natalia Gromak .

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Editors and Affiliations

  1. CABIMER, Universidad de Sevilla, Seville, Spain

    Andrés Aguilera

  2. Skryabin Institute of Bioengineering, Research Centre of Biotechnology RAS, Moscow, Russia

    Alexey Ruzov

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Beghè, C., Gromak, N. (2022). R-Loop Immunoprecipitation: A Method to Detect R-Loop Interacting Factors . In: Aguilera, A., Ruzov, A. (eds) R-Loops . Methods in Molecular Biology, vol 2528. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2477-7_14

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  • DOI: https://doi.org/10.1007/978-1-0716-2477-7_14

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