iMUT-seq: high-resolution mapping of DSB mutational landscapes reveals new insights into the mutagenic mechanisms of DSB repair

DNA double-strand breaks (DSBs) are the most mutagenic form of DNA damage, and play a significant role in cancer biology, neurodegeneration and aging. However, studying DSB-induced mutagenesis is currently limited by the tools available for mapping these mutations. Here, we describe iMUT-seq, a technique that profiles DSB-induced mutations at high-sensitivity and single-nucleotide resolution around endogenous DSBs spread across the genome. By depleting 20 different DSB-repair factors we defined their mutational signatures in detail, revealing remarkable insights into the mechanisms of DSB-induced mutagenesis. We find that homologous-recombination (HR) is mutagenic in nature, displaying high levels of base substitutions and mononucleotide deletions due to DNA-polymerase errors, but simultaneously reduced translocation events, suggesting the primary role of HR is the specific suppression of genomic rearrangements. The results presented here offer new fundamental insights into DSB-induced mutagenesis and have significant implications for our understanding of cancer biology and the development of DDR-targeting chemotherapeutics.