Repair of Multiple Simultaneous Double-Strand Breaks Causes Genome-wide Formation of Long Single Strand DNA and Bursts of Clustered Hypermutation

A single cancer genome can harbor thousands of clustered mutations. Mutation signature analyses have revealed that the origin of clusters are lesions in long tracts of single-strand (ss) DNA damaged by APOBEC cytidine deaminases, raising questions about molecular mechanisms that generate long ssDNA vulnerable to hypermutation. Here, we show that ssDNA-intermediates formed during the repair of gamma-induced bursts of double-strand breaks (DSBs) in the presence of APOBEC3A in yeast lead to multiple APOBEC-induced clusters similar to cancer. We identified three independent pathways enabling cluster formation associated with repairing bursts of DSBs: 5’-3’ bi-directional resection, uni-directional resection and break-induced replication (BIR). Analysis of millions of mutations in APOBEC hypermutated cancer genomes, revealed that cancer tolerance to formation of hypermutable ssDNA is similar to yeast and that the predominant pattern of clustered mutagenesis is the same as in resection-defective yeast, suggesting that cluster formation in cancers is driven by a BIR-like mechanism.