Bisulfite treatment and single-molecule real-time sequencing reveal d-loop length, position, and distribution

Shanaya Shital Shah, Stella R. Hartono, Frédéric Chédin, Wolf-Dietrich Heyer

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

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. Nondenaturing 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 realtime 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.

Original languageEnglish (US)
Article numbere59111
Pages (from-to)1-25
Number of pages25
JournaleLife
Volume9
DOIs
StatePublished - Oct 2020

ASJC Scopus subject areas

  • Neuroscience(all)
  • Immunology and Microbiology(all)
  • Biochemistry, Genetics and Molecular Biology(all)

Fingerprint

Dive into the research topics of 'Bisulfite treatment and single-molecule real-time sequencing reveal d-loop length, position, and distribution'. Together they form a unique fingerprint.

Cite this