Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing

作者：Crosetto, Nicola; Mitra, Abhishek; Silva, Maria Joao; Bienko, Magda; Dojer, Norbert; Wang, Qi; Karaca, Elif; Chiarle, Roberto; Skrzypczak, Magdalena; Ginalski, Krzysztof; Pasero, Philippe; Rowicka, Maga^*; Dikic, Ivan

来源：Nature Methods, 2013, 10(4): 361-+.

DOI：10.1038/nmeth.2408

• CLASS SWITCH RECOMBINATION
• HUMAN CANCER GENES
• H2AX PHOSPHORYLATION
• GENOMIC INSTABILITY
• DAMAGE RESPONSE
• FRAGILE SITES
• REPLICATION
• GAMMA-H2AX
• CHROMOSOME
• CELLS

We present a genome-wide approach to map DNA double-strand breaks (DSBs) at nucleotide resolution by a method we termed BLESS (direct in situ breaks labeling, enrichment on streptavidin and next-generation sequencing). We validated and tested BLESS using human and mouse cells and different DSBs-inducing agents and sequencing platforms. BLESS was able to detect telomere ends, Sce endonuclease-induced DSBs and complex genome-wide DSB landscapes. As a proof of principle, we characterized the genomic landscape of sensitivity to replication stress in human cells, and we identified > 2,000 nonuniformly distributed aphidicolin-sensitive regions (ASRs) overrepresented in genes and enriched in satellite repeats. ASRs were also enriched in regions rearranged in human cancers, with many cancer-associated genes exhibiting high sensitivity to replication stress. Our method is suitable for genome-wide mapping of DSBs in various cells and experimental conditions, with a specificity and resolution unachievable by current techniques.

• 出版日期2013-4