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
Mentions: To confirm the inactivation of replication forks by long HU blocks, we measured the formation of the phosphorylated histone variant H2AX (γH2AX) at inactivated forks. γH2AX accumulates quickly during HU blocks (Figures 2A and 2B), even before DSB induction is observed (Figure 2C) (Saintigny et al., 2001). The γH2AX signal colocalized with RPA foci, suggesting that it marks regions of extensive single-stranded DNA at stalled forks (Figure 2A). We released cells from the HU block for 1 hr and measured how much γH2AX remained after replication had resumed (Figure 2B). We found that γH2AX rapidly disappeared after release from the 2 hr HU block. In contrast, γH2AX foci persisted after release from 24 hr HU block, at times when more DSB were induced (Figure 2B, C). Persisting γH2AX foci colocalized with stalled or inactivated replication forks (Figure 2D). These observations show that DNA damage accumulates at stalled forks with increasing lengths of HU treatments and that this DNA damage persists in cells released from long HU blocks.
Affiliation: Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford OX3 7DQ, UK.