Unrepaired base excision repair intermediates in template DNA strands trigger replication fork collapse and PARP inhibitor sensitivity
DNA single-strand breaks (SSBs) interrupt DNA replication and lead to chromosome breakage. However, it’s uncertain whether SSBs cause chromosome breakage when located behind or ahead of replication forks. To explore this, we utilized the heightened sensitivity of SSB repair-deficient human cells lacking PARP activity or XRCC1 to the thymidine analogue 5-chloro-2′-deoxyuridine (CldU). Our findings reveal that treating these cells with CldU results in chromosome breakage, sister chromatid exchange, and cytotoxicity through a mechanism that depends on the S phase activity of uracil DNA glycosylase (UNG). Notably, we demonstrate that CldU incorporation during one cell cycle is cytotoxic only in the subsequent cell cycle when it is present in the template DNA. Supporting this, although UNG induces SSBs in nascent strands behind replication forks and template strands ahead of replication forks, only the SSBs in the latter lead to fork collapse and chromosome breakage. Lastly, we show that BRCA-deficient cells are highly sensitive to CldU, either alone or in combination with a PARP inhibitor, indicating potential clinical applications for CldU.