Bisulfite treatment and single-molecule real-time sequencing reveals D-loop length, position and distribution

  1. Shanaya Shital Shah
  2. Stella R Hartono
  3. Frédéric Chédin
  4. Wolf-Dietrich Heyer  Is a corresponding author
  1. University of California, Davis, United States

Abstract

Displacement loops (D-loops) are signature intermediates formed during homologous recombination. Numerous factors regulate D-loop formation and disruption, thereby influencing crucial aspects of DNA repair, including donor choice and the possibility of crossover outcome. While D-loop detection methods exist, it is currently unfeasible to assess the relationship between D-loop editors and D-loop characteristics such as length and position. Here, we developed a novel in vitro assay to characterize the length and position of individual D-loops with near base-pair resolution and deep coverage, while also revealing their distribution in a population. Non-denaturing bisulfite treatment modifies the cytosines on the displaced strand of the D-loop to uracil, leaving a permanent signature for the displaced strand. Subsequent single-molecule real-time sequencing uncovers the cytosine conversion patch as a D-loop footprint. The D-loop Mapping Assay is widely applicable with different substrates and donor types and can be used to study factors that influence D-loop properties.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for all numerical data.

Article and author information

Author details

  1. Shanaya Shital Shah

    Microbiology and Molecular Genetics, University of California, Davis, Davis, United States
    Competing interests
    No competing interests declared.
  2. Stella R Hartono

    Department of Molecular and Cellular Biology, University of California, Davis, Davis, United States
    Competing interests
    No competing interests declared.
  3. Frédéric Chédin

    Department of Molecular and Cellular Biology, University of California, Davis, Davis, United States
    Competing interests
    No competing interests declared.
  4. Wolf-Dietrich Heyer

    Microbiology and Molecular Genetics, University of California, Davis, Davis, United States
    For correspondence
    wdHeyer@ucdavis.edu
    Competing interests
    Wolf-Dietrich Heyer, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7774-1953

Funding

National Institutes of Health (GM 58015)

  • Wolf-Dietrich Heyer

National Institutes of Health (CA 92276)

  • Wolf-Dietrich Heyer

National Institutes of Health (GM 120607)

  • Frédéric Chédin

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Maria Spies, University of Iowa, United States

Publication history

  1. Received: May 20, 2020
  2. Accepted: October 27, 2020
  3. Accepted Manuscript published: November 13, 2020 (version 1)
  4. Version of Record published: November 27, 2020 (version 2)

Copyright

© 2020, Shah et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Shanaya Shital Shah
  2. Stella R Hartono
  3. Frédéric Chédin
  4. Wolf-Dietrich Heyer
(2020)
Bisulfite treatment and single-molecule real-time sequencing reveals D-loop length, position and distribution
eLife 9:e59111.
https://doi.org/10.7554/eLife.59111
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