Hydroxyurea-stalled replication forks become progressively inactivated and require two different RAD51-mediated pathways for restart and repair.
Bottom Line: Hydroxyurea (HU) depletes the cells of dNTPs, which initially results in stalled replication forks that, after prolonged treatment, collapse into DSBs.Here, we report that stalled replication forks are efficiently restarted in a RAD51-dependent process that does not trigger homologous recombination (HR).In contrast, replication forks collapsed by prolonged replication blocks do not restart, and global replication is rescued by new origin firing.
Affiliation: Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford OX3 7DQ, UK.Show MeSH
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Mentions: Although reduced fork restart after long HU blocks was accompanied by increased new origin firing, origin firing was not elevated when RAD51-depled cells were released from short HU blocks (Figure 6A). To test whether this might be due to origin suppression by the S phase checkpoint, we analyzed replication restart in the presence or absence of the Chk1 inhibitor CEP-3891 (Figure 6B). We found that Chk1 inhibition alone did reduce fork restart to the same extent as RAD51 depletion, which is in agreement with previous reports that Chk1 stabilizes stalled replication forks (Feijoo et al., 2001; Zachos et al., 2003). Chk1 inhibition also dramatically increased new origin firing after release from 2 hr HU block (Figure 6B). These effects were specific to HU treatment (Figure S3). When RAD51 depletion and Chk1 inhibition were combined, we observed an additive effect on both fork stalling and new origin firing (Figure 6B). This finding suggests that failed fork restart due to RAD51 depletion can trigger new origin firing, but after short HU blocks, the checkpoint prevents this from being a significant mechanism of replication restart. Even after release from 24 hr HU treatment, when new origin firing was increased by 10 fold, Chk1 inhibition during the time of restart increased new origin firing even further (Figure 6C), suggesting that the S phase checkpoint still suppresses a large amount of origin firing after release from long HU blocks. Chk1 inhibitor was only present during the last hour of HU block and did not affect fork restart (Figure 6C).
Affiliation: Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford OX3 7DQ, UK.