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.

中文翻译：

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非破坏性亚硫酸氢盐处理和单分子实时排序显示D回路脚印，长度，位置和分布

置换环（D-loop）是在同源重组过程中形成的标志性中间体。许多因素调节D环的形成和破坏，从而影响DNA修复的关键方面，包括供体的选择和交叉结果的可能性。尽管存在D回路检测方法，但目前尚无法评估D回路编辑器与D回路特性（例如长度和位置）之间的关系。在这里，我们开发了一种新颖的体外测定法，以碱基对分辨率和深度覆盖来表征单个D环的长度和位置，同时还揭示了它们在人群中的分布。非变性亚硫酸氢盐处理将D环置换链上的胞嘧啶修饰为尿嘧啶，为置换链留下永久性标记。随后的单分子实时测序揭示了胞嘧啶转化斑作为D环足迹，以前所未有的分辨率揭示了D环特征。D环作图分析广泛适用于不同的底物和供体类型，可用于研究影响D环特性的因素。