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Hydroxyurea-stalled replication forks become progressively inactivated and require two different RAD51-mediated pathways for restart and repair.

Petermann E, Orta ML, Issaeva N, Schultz N, Helleday T - Mol. Cell (2010)

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.

View Article: PubMed Central - PubMed

Affiliation: Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford OX3 7DQ, UK.

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After Long Replication Blocks, RAD51 Foci Do Not Promote Fork Restart but Are Required for DNA Damage Repair(A) Percentage of control- or RAD51-depleted U2OS cells containing more than 10 RAD51 foci after 24 hr treatment with 0.5 mM HU. The means and range (bars) of two independent experiments are shown. Values marked with asterisks are significantly different from control (p < 0.01).(B) Fork restart and new origin firing after release from 24 hr treatment with 2 mM HU in control- or RAD51-depleted U2OS cells. The means and SD (bars) of three independent experiments are shown. Replication structures are shown as percentage of all CldU-labeled tracks.(C) Pulsed-field gel electrophoresis to visualize DSB remaining in control- or RAD51-depleted U2OS cells after 0, 12 24, 36, and 48 hr release from 24 hr treatment with 2 mM HU.(D) Quantification of DSB remaining in control- or RAD51-depleted U2OS cells as in (C). The means and range (bars) of two to three independent experiments are shown.(E) Model of RAD51-mediated replication fork restart and repair. RAD51 may have a similar role as recA in E. coli, promoting the formation of a Holliday Junction intermediate (chicken foot). The DNA end may then assist to restart replication that would involve recombination over a small area (short tract). Holliday Junction dissolution by the BLM-Top3 complex would dissolve any remaining double Holliday Junctions (Wu and Hickson, 2003). New origin firing rescues replication of collapsed replication forks, which are then repaired by long tract HR involving double Holliday Junction dissolution, synthesis-dependent strand annealing (SDSA) or nonhomologous end joining (see also Figure S4).
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fig7: After Long Replication Blocks, RAD51 Foci Do Not Promote Fork Restart but Are Required for DNA Damage Repair(A) Percentage of control- or RAD51-depleted U2OS cells containing more than 10 RAD51 foci after 24 hr treatment with 0.5 mM HU. The means and range (bars) of two independent experiments are shown. Values marked with asterisks are significantly different from control (p < 0.01).(B) Fork restart and new origin firing after release from 24 hr treatment with 2 mM HU in control- or RAD51-depleted U2OS cells. The means and SD (bars) of three independent experiments are shown. Replication structures are shown as percentage of all CldU-labeled tracks.(C) Pulsed-field gel electrophoresis to visualize DSB remaining in control- or RAD51-depleted U2OS cells after 0, 12 24, 36, and 48 hr release from 24 hr treatment with 2 mM HU.(D) Quantification of DSB remaining in control- or RAD51-depleted U2OS cells as in (C). The means and range (bars) of two to three independent experiments are shown.(E) Model of RAD51-mediated replication fork restart and repair. RAD51 may have a similar role as recA in E. coli, promoting the formation of a Holliday Junction intermediate (chicken foot). The DNA end may then assist to restart replication that would involve recombination over a small area (short tract). Holliday Junction dissolution by the BLM-Top3 complex would dissolve any remaining double Holliday Junctions (Wu and Hickson, 2003). New origin firing rescues replication of collapsed replication forks, which are then repaired by long tract HR involving double Holliday Junction dissolution, synthesis-dependent strand annealing (SDSA) or nonhomologous end joining (see also Figure S4).

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.


Hydroxyurea-stalled replication forks become progressively inactivated and require two different RAD51-mediated pathways for restart and repair.

Petermann E, Orta ML, Issaeva N, Schultz N, Helleday T - Mol. Cell (2010)

After Long Replication Blocks, RAD51 Foci Do Not Promote Fork Restart but Are Required for DNA Damage Repair(A) Percentage of control- or RAD51-depleted U2OS cells containing more than 10 RAD51 foci after 24 hr treatment with 0.5 mM HU. The means and range (bars) of two independent experiments are shown. Values marked with asterisks are significantly different from control (p < 0.01).(B) Fork restart and new origin firing after release from 24 hr treatment with 2 mM HU in control- or RAD51-depleted U2OS cells. The means and SD (bars) of three independent experiments are shown. Replication structures are shown as percentage of all CldU-labeled tracks.(C) Pulsed-field gel electrophoresis to visualize DSB remaining in control- or RAD51-depleted U2OS cells after 0, 12 24, 36, and 48 hr release from 24 hr treatment with 2 mM HU.(D) Quantification of DSB remaining in control- or RAD51-depleted U2OS cells as in (C). The means and range (bars) of two to three independent experiments are shown.(E) Model of RAD51-mediated replication fork restart and repair. RAD51 may have a similar role as recA in E. coli, promoting the formation of a Holliday Junction intermediate (chicken foot). The DNA end may then assist to restart replication that would involve recombination over a small area (short tract). Holliday Junction dissolution by the BLM-Top3 complex would dissolve any remaining double Holliday Junctions (Wu and Hickson, 2003). New origin firing rescues replication of collapsed replication forks, which are then repaired by long tract HR involving double Holliday Junction dissolution, synthesis-dependent strand annealing (SDSA) or nonhomologous end joining (see also Figure S4).
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fig7: After Long Replication Blocks, RAD51 Foci Do Not Promote Fork Restart but Are Required for DNA Damage Repair(A) Percentage of control- or RAD51-depleted U2OS cells containing more than 10 RAD51 foci after 24 hr treatment with 0.5 mM HU. The means and range (bars) of two independent experiments are shown. Values marked with asterisks are significantly different from control (p < 0.01).(B) Fork restart and new origin firing after release from 24 hr treatment with 2 mM HU in control- or RAD51-depleted U2OS cells. The means and SD (bars) of three independent experiments are shown. Replication structures are shown as percentage of all CldU-labeled tracks.(C) Pulsed-field gel electrophoresis to visualize DSB remaining in control- or RAD51-depleted U2OS cells after 0, 12 24, 36, and 48 hr release from 24 hr treatment with 2 mM HU.(D) Quantification of DSB remaining in control- or RAD51-depleted U2OS cells as in (C). The means and range (bars) of two to three independent experiments are shown.(E) Model of RAD51-mediated replication fork restart and repair. RAD51 may have a similar role as recA in E. coli, promoting the formation of a Holliday Junction intermediate (chicken foot). The DNA end may then assist to restart replication that would involve recombination over a small area (short tract). Holliday Junction dissolution by the BLM-Top3 complex would dissolve any remaining double Holliday Junctions (Wu and Hickson, 2003). New origin firing rescues replication of collapsed replication forks, which are then repaired by long tract HR involving double Holliday Junction dissolution, synthesis-dependent strand annealing (SDSA) or nonhomologous end joining (see also Figure S4).
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.

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.

View Article: PubMed Central - PubMed

Affiliation: Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford OX3 7DQ, UK.

Show MeSH