Problems during DNA replication often cause chromosomal abnormalities when replication forks become unstable and fail to progress properly. If forks stall, mechanisms become activated to maintain replication fork stability, restart progression and avoid collapse into double-strand DNA breaks (DSBs). ATR (ataxia telangiectasia and rad3-related) is thought to play an important role in maintaining replication fork stability, as evidenced by a dramatic increase in DSBs upon stalled DNA replication in ATR-deficient cells.;Here we demonstrate that, upon stalled replication, ATR-deficient cells have increased H2AX phosphorylation and Rad51 foci, markers of DSB formation. The ATR-related kinases, ATM and DNA-PKcs, mediate this phosphorylation. Depletion of ATR in H2AX-deficient cells leads to increased DSBs in metaphase spreads. A significant decrease in Rad51 foci was seen in ATR knockout cells when H2AX was absent, suggesting a failure of homologous recombination-mediated repair mechanisms in the absence of H2AX. Consistent with a failure of normal repair mechanisms, sister chromatid translocation events increase in ATR/H2AX double knockout cells, suggesting that ATR and H2AX work cooperatively to suppress DSBs upon stalled DNA replication.
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