NON-DENATURING BISULFITE TREATMENT AND SINGLE-MOLECULE REAL-TIME SEQUENCING REVEALS D-LOOP FOOTPRINTS, THEIR LENGTH, POSITION AND DISTRIBUTION

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 a 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-loop with 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, revealing D-loop characteristics at unprecedented resolution. 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.