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

DNA double-stranded breaks (DSBs) are an especially toxic form of damage that can be reversed without loss of genetic information by homology-directed repair (HDR), which copies 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. A major challenge in understanding homology search is the lack of tools to monitor search itself, rather than the end-stage outcome of HDR. We developed RAD51 proximity identification sequencing (RaPID-seq), a highly sensitive in cellulo method that marks all DNA searched by RAD51, regardless of whether it is chosen as the final template. We find that HDR in human cells is hierarchical. Only DSB-proximal sequences are searched, and even identical sequences outside the proximal window are not efficiently interrogated. Final recombination is unlocked by template homology but only at proximal sequences. We further find that RAD51 efficiently searches exogenously introduced DNA templates, such as those commonly used during genome editing, even those with no homology to the DSB site. Such highly abundant exogenous templates compete with search of endogenous genome templates. Our data reveal the invisible process of homology search and shed new light on fundamental mechanisms underlying genome editing.