HCoDES Reveals Chromosomal DNA End Structures with Single-Nucleotide Resolution

Summary The structure of broken DNA endsis a critical determinant of the pathway used for DNA double-strand break (DSB) repair. Here, we develop an approach involving the hairpin capture of DNA endstructures (HCoDES), which elucidates chromosomal DNA endstructures at single-nucleotide resolution. HCoDES defines structures of physiologic DSBs generated by the RAG endonuclease, as well as those generated by nucleases widely used for genome editing. Analysis of G1 phasecells deficient in H2AX or 53BP1 reveals DNA endsthat are frequently resected to form long single-stranded overhangs that can be repaired by mutagenic pathways. In addition to 3′ overhangs, many of these DNA endsunexpectedly form long 5′ single-stranded overhangs. The divergence in DNA endstructures resolved by HCoDES suggests that H2AX and 53BP1 may have distinct activities in endprotection. Thus, the high-resolution endstructures obtained by HCoDES identify features of DNA endprocessing during DSB repair.