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: Our data suggested that RAD51 promotes replication fork restart after short replication blocks, when forks are still competent to restart. Next, we wanted to analyze the effect of RAD51 on fork restart after long HU blocks, when RAD51 foci accumulate and HR is activated. We confirmed that control cells, but not RAD51-depleted cells, formed RAD51 foci in response to 24 hr HU block (Figure 7A). We used the DNA fiber technique to determine the effect of RAD51 depletion and lack of RAD51 foci on fork restart after 24 hr HU block. We found that, in contrast to release from short HU treatments, RAD51 depletion does not decrease the number of forks that restart after release from long HU blocks (Figure 7B). RAD51 depletion did not affect new origin firing (Figure 7B). However RAD51-depleted cells repair DNA damage induced by long HU treatment less efficiently than do control cells, as demonstrated by the higher amounts of DSB remaining in RAD51-depleted cells up to 48 hr after release from HU block (Figures 7C and 7D). Similarly, a larger number of RAD51-depleted cells still contained γH2AX signal up to 48 hr after release from HU (Figure S4). These observations suggest that there is no correlation between the ability of cells to form RAD51 foci and the ability to restart replication forks. Rather, RAD51 foci formation coincides with replication fork inactivation, DSB formation, and the requirement for RAD51 for DNA repair. Taken together, these data support the idea that RAD51 protein promotes fork restart without forming foci, whereas RAD51 foci formation is a step in the recombination process that repairs collapsed forks.
Affiliation: Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford OX3 7DQ, UK.