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Analysis of protein-DNA interactions in chromatin by UV induced cross-linking and mass spectrometry

Author

Listed:
  • Alexandra Stützer

    (Max Planck Institute for Biophysical Chemistry)

  • Luisa M. Welp

    (Max Planck Institute for Biophysical Chemistry)

  • Monika Raabe

    (Max Planck Institute for Biophysical Chemistry)

  • Timo Sachsenberg

    (University of Tübingen
    University of Tübingen)

  • Christin Kappert

    (Max Planck Institute for Biophysical Chemistry
    Max Planck Institute of Experimental Medicine)

  • Alexander Wulf

    (Max Planck Institute for Biophysical Chemistry)

  • Andy M. Lau

    (King’s College London)

  • Stefan-Sebastian David

    (Max Planck Institute for Biophysical Chemistry
    Laboratory of Chromatin Biochemistry)

  • Aleksandar Chernev

    (Max Planck Institute for Biophysical Chemistry)

  • Katharina Kramer

    (CSL Behring GmbH)

  • Argyris Politis

    (King’s College London)

  • Oliver Kohlbacher

    (University of Tübingen
    University of Tübingen
    University Hospital Tübingen
    Max Planck Institute for Developmental Biology)

  • Wolfgang Fischle

    (Max Planck Institute for Biophysical Chemistry
    Laboratory of Chromatin Biochemistry)

  • Henning Urlaub

    (Max Planck Institute for Biophysical Chemistry
    University Medical Center Göttingen)

Abstract

Protein–DNA interactions are key to the functionality and stability of the genome. Identification and mapping of protein–DNA interaction interfaces and sites is crucial for understanding DNA-dependent processes. Here, we present a workflow that allows mass spectrometric (MS) identification of proteins in direct contact with DNA in reconstituted and native chromatin after cross-linking by ultraviolet (UV) light. Our approach enables the determination of contact interfaces at amino-acid level. With the example of chromatin-associated protein SCML2 we show that our technique allows differentiation of nucleosome-binding interfaces in distinct states. By UV cross-linking of isolated nuclei we determined the cross-linking sites of several factors including chromatin-modifying enzymes, demonstrating that our workflow is not restricted to reconstituted materials. As our approach can distinguish between protein–RNA and DNA interactions in one single experiment, we project that it will be possible to obtain insights into chromatin and its regulation in the future.

Suggested Citation

  • Alexandra Stützer & Luisa M. Welp & Monika Raabe & Timo Sachsenberg & Christin Kappert & Alexander Wulf & Andy M. Lau & Stefan-Sebastian David & Aleksandar Chernev & Katharina Kramer & Argyris Politis, 2020. "Analysis of protein-DNA interactions in chromatin by UV induced cross-linking and mass spectrometry," Nature Communications, Nature, vol. 11(1), pages 1-12, December.
    • Handle: RePEc:nat:natcom:v:11:y:2020:i:1:d:10.1038_s41467-020-19047-7
    DOI: 10.1038/s41467-020-19047-7
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    Cited by:

    1. Jong Woo Bae & Sangtae Kim & V. Narry Kim & Jong-Seo Kim, 2021. "Photoactivatable ribonucleosides mark base-specific RNA-binding sites," Nature Communications, Nature, vol. 12(1), pages 1-10, December.

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