HCoDES Reveals Chromosomal DNA End Structures with Single-Nucleotide Resolution

• Published in: Molecular cell Vol. 56; no. 6; pp. 808 - 818
• Main Authors: Dorsett, Yair, Zhou, Yanjiao, Tubbs, Anthony T., Chen, Bo-Ruei, Purman, Caitlin, Lee, Baeck-Seung, George, Rosmy, Bredemeyer, Andrea L., Zhao, Jiang-yang, Sodergen, Erica, Weinstock, George M., Han, Nathan D., Reyes, Alejandro, Oltz, Eugene M., Dorsett, Dale, Misulovin, Ziva, Payton, Jacqueline E., Sleckman, Barry P.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 18.12.2014
• Subjects: Base Sequence; Cells, Cultured; Chromosomes, Human - genetics; DNA Breaks, Double-Stranded; Humans; Sequence Analysis, DNA - methods
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2014.10.024

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 involving the 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 features of DNA end processing during DSB repair. [Display omitted] •HCoDES assay reveals chromosomal DNA end structures at single-nucleotide resolution•H2AX and 53BP1 prevent single-stranded DNA overhangs in G1 phase cells•H2AX and 53BP1 may have distinct functions in DNA end protection•Aberrant DNA end resection can generate de novo double-stranded DNA breaks Dorsett et al. describe a method (HCoDES) that allows for the determination of chromosomal DNA end structures at single-nucleotide resolution. DNA end structures are examined in G1 phase cells. Their findings establish the broad utility of HCoDES and reveal functions for H2AX and 53BP1 in DNA end protection.