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