Unrepaired base excision repair intermediates in template DNA strands trigger replication fork collapse and PARP inhibitor sensitivity

Abstract

DNA single‐strand breaks (SSBs) disrupt DNA replication and induce chromosome breakage. However, whether SSBs induce chromosome breakage when present behind replication forks or ahead of replication forks is unclear. To address this question, we exploited an exquisite sensitivity of SSB repair‐defective human cells lacking PARP activity or XRCC1 to the thymidine analogue 5‐chloro‐2′‐deoxyuridine (CldU). We show that incubation with CldU in these cells results in chromosome breakage, sister chromatid exchange, and cytotoxicity by a mechanism that depends on the S phase activity of uracil DNA glycosylase (UNG). Importantly, we show that CldU incorporation in one cell cycle is cytotoxic only during the following cell cycle, when it is present in template DNA. In agreement with this, while UNG induces SSBs both in nascent strands behind replication forks and in template strands ahead of replication forks, only the latter trigger fork collapse and chromosome breakage. Finally, we show that BRCA‐defective cells are hypersensitive to CldU, either alone and/or in combination with PARP inhibitor, suggesting that CldU may have clinical utility. image UNG glycosylase excises genomic chlorouracil from cells treated with 5‐chloro‐2′‐deoxyuridine (CldU), inducing template strand breaks and replication fork collapse. Cells lacking single‐strand break repair or BRCA1/BRCA2 are hypersensitive to CldU, alone and in combination with PARP inhibitor. The DNA glycosylase UNG excises chlorouracil, inducing DNA single‐strand breaks that collapse DNA replication forks. Human cells lacking the single‐strand break repair protein XRCC1 or incubated with PARP inhibitor are exquisitely sensitive to the thymine analogue, 5‐chloro‐2′‐deoxyuridine (CldU). CldU may have clinical utility for BRCA‐defective cancers, alone or in combination with PARP inhibitors.