GSE62534
Hairpin Capture of DNA End Structures reveals chromosomal DNA end structure with single nucleotide resolution
The structure of broken DNA ends is a critical determinant of the pathway used for DNA double strand break (DSB) repair.  Here, we develop an approach, hairpin capture of DNA end structures (HCoDES), which elucidates chromosomal DNA end structures 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-phase cells deficient in H2AX or 53BP1 reveals DNA ends that 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 ends unexpectedly form long 5’ single-stranded overhangs.  The divergence in DNA end structures resolved by HCoDES suggests that H2AX and 53BP1 may have distinct activities in end protection.  Thus, the high-resolution end structures obtained by HCoDES identify new features of DNA end processing during DSB repair. Overall design: Single stranded DNA ligation of genomic DNA isolated from G1 arrested LigaseIV-/-, LigaseIV-/- 53BP1-/- and LigaseIV-/- H2AX-/- Abelson pre-B cells harboring site specific DSBs generated by the RAG recombinase, Cas9 endonuclease or Zinc Finger Endonuclease.