RAD51 proximity mapping reveals spatial constraints on homology search during DNA double-stranded break repair

DNA double-stranded breaks (DSBs) are toxic events that can be reversed without genetic information loss by homology-directed repair (HDR), wherein information is copied from an intact template molecule. Finding a correct template within millions to billions of other DNA bases is termed homology search and is mediated by the protein RAD51. To monitor transient search in cellulo, we develop RAD51 proximity identification sequencing (RaPID-seq), a highly sensitive method marking all DNA searched regardless of whether it is chosen as the final template. We find that HDR in human cells is hierarchical with DSB proximity constraining the search space from which sequence homology determines the chosen template. Exogenously introduced DNA templates, such as those used during genome editing, are unconstrained and efficiently searched by the DSB, thereby competing with endogenous template search. Our data reveal the invisible process of homology search and shed new light on fundamental mechanisms underlying genome editing.