1 Citation
DNA single-strand breaks (SSBs) disrupt DNA replication and induce
chromosome breakage. However, whether SSBs induce chromosome breakage when
present in nascent strands behind replication forks or template strands
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 analog
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 triggers 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.