Clastogenesis by low densities of nucleotide lesions requires the completion of two cell cycles

Damaged DNA nucleotides can trigger genome rearrangements through clastogenesis, a process driven by erroneous repair of double-strand breaks (DSBs) and associated with cancer development. While DSBs are known to arise from endonuclease activity at stalled replication forks, their clastogenic potential has remained uncertain. Here, we have used wild-type, nucleotide excision repair (NER)-deficient and translesion synthesis (TLS)-deficient cells, combined with advanced cytogenetic analyses, to identify a previously unrecognized mechanism of clastogenesis. We demonstrate that, Rpa-protected single-stranded DNA (ssDNA) tracts harboring unrepaired lesions can persist through mitosis. Only during the subsequent S phase, these tracts are converted into, highly clastogenic, DSBs. Consistent with a role of this mechanism in carcinogenesis, prostate cancers exhibiting extensive genomic rearrangements frequently harbor somatic defects in NER or in error-free homologous recombination-mediated DSB repair. These findings provide critical mechanistic insight and highlight potential implications for routine clastogenicity testing.