TY - JOUR
T1 - Bisulfite treatment and single-molecule real-time sequencing reveal d-loop length, position, and distribution
AU - Shah, Shanaya Shital
AU - Hartono, Stella R.
AU - Chédin, Frédéric
AU - Heyer, Wolf-Dietrich
N1 - Funding Information:
We thank members of the Heyer laboratory, especially Aurele Piazza and William Wright, for stimulating discussions. We thank Diedre Reitz for helpful feedback on the manuscript. We also thank members of the Chedin laboratory, in particular, Lionel Sanz, for providing AMPure beads and Maika Malig for technical help. We also thank the DNA core technologies at UC Davis and the UC Berkeley Genomics Facility for providing PacBio sequencing services. This research used core services supported by P30 CA93373 and was supported by NIH grants GM58015 and CA92276 to W.-D.H. and NIH grant GM120607 to FC.
PY - 2020/10
Y1 - 2020/10
N2 - 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.
AB - 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.
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U2 - 10.7554/eLife.59111
DO - 10.7554/eLife.59111
M3 - Article
C2 - 33185185
AN - SCOPUS:85097003967
VL - 9
SP - 1
EP - 25
JO - eLife
JF - eLife
SN - 2050-084X
M1 - e59111
ER -