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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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Late Activation of Homologous Recombination during HU Block(A) HU-induced RAD51 foci in U2OS cells. Cells were left untreated or treated with 2 mM HU for 1, 2, or 24 hr, fixed, and immunostained for RAD51 (red). DNA was counterstained with DAPI (blue).(B) Quantification of HU-induced RAD51 foci in U2OS cells. Cells were treated with 2 mM HU for the times indicated and immunostained for RAD51. Cells containing >10 RAD51 foci were scored as positive. The means and range (bars) of two independent experiments are shown.(C) Recombination frequencies in SPD8 cells induced by HU. Cells were treated with 2 mM HU for the times indicated and recovered for 48 hr. HPRT+ revertants were quantified 7 days later. The means and SD (bars) of four to ten independent experiments are shown. Values marked with asterisks are significantly different from control (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; see also Figure S2).
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fig3: Late Activation of Homologous Recombination during HU Block(A) HU-induced RAD51 foci in U2OS cells. Cells were left untreated or treated with 2 mM HU for 1, 2, or 24 hr, fixed, and immunostained for RAD51 (red). DNA was counterstained with DAPI (blue).(B) Quantification of HU-induced RAD51 foci in U2OS cells. Cells were treated with 2 mM HU for the times indicated and immunostained for RAD51. Cells containing >10 RAD51 foci were scored as positive. The means and range (bars) of two independent experiments are shown.(C) Recombination frequencies in SPD8 cells induced by HU. Cells were treated with 2 mM HU for the times indicated and recovered for 48 hr. HPRT+ revertants were quantified 7 days later. The means and SD (bars) of four to ten independent experiments are shown. Values marked with asterisks are significantly different from control (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; see also Figure S2).

Mentions: To analyze the response of RAD51 and HR at early and late times of replication blocks, we performed time courses of RAD51 foci formation and HR frequencies induced by HU treatment. Cells were treated with HU for different times, and the percentages of cells containing more than 10 RAD51 foci were quantified. RAD51 foci are induced after 24 hr, but not after 1 or 2 hr HU treatment in U2OS cells (Figures 3A and 3B). To analyze the time course of HR induction by HU treatments, we used the SPD8 cell line, which carries a recombination reporter in the hprt gene. HR by either unequal sister chromatid exchange, intrachromatid exchange, single-strand annealing, or gene conversion can lead to restoration of the wild-type hprt gene encoding a functional HGPRT protein (Helleday et al., 1998). We found that HR is induced by HU treatments of 24, but not by treatments of 1 or 2 hr (Figure 3C). This finding suggested that HR is not active while replication forks restart, but is induced when replication forks become inactivated. Because we used SPD8 cells to measure HR, we confirmed that they display similar progressive replication fork inactivation as do U2OS cells (Figures S2A–S2D). Forks become inactivated considerably earlier in SPD8 cells, accompanied by earlier RAD51 foci formation (Figure S2E), which colocalize with stalled replication forks (Figure S2F). DSB induction occurred earlier in SPD8 cells as well, although there was no difference to U2OS cells at 2 hr (Figure S2G). These observations confirm that HR activation occurs late during replication blocks and coincides with or is preceded by replication fork inactivation and DSB formation.


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)

Late Activation of Homologous Recombination during HU Block(A) HU-induced RAD51 foci in U2OS cells. Cells were left untreated or treated with 2 mM HU for 1, 2, or 24 hr, fixed, and immunostained for RAD51 (red). DNA was counterstained with DAPI (blue).(B) Quantification of HU-induced RAD51 foci in U2OS cells. Cells were treated with 2 mM HU for the times indicated and immunostained for RAD51. Cells containing >10 RAD51 foci were scored as positive. The means and range (bars) of two independent experiments are shown.(C) Recombination frequencies in SPD8 cells induced by HU. Cells were treated with 2 mM HU for the times indicated and recovered for 48 hr. HPRT+ revertants were quantified 7 days later. The means and SD (bars) of four to ten independent experiments are shown. Values marked with asterisks are significantly different from control (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; see also Figure S2).
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fig3: Late Activation of Homologous Recombination during HU Block(A) HU-induced RAD51 foci in U2OS cells. Cells were left untreated or treated with 2 mM HU for 1, 2, or 24 hr, fixed, and immunostained for RAD51 (red). DNA was counterstained with DAPI (blue).(B) Quantification of HU-induced RAD51 foci in U2OS cells. Cells were treated with 2 mM HU for the times indicated and immunostained for RAD51. Cells containing >10 RAD51 foci were scored as positive. The means and range (bars) of two independent experiments are shown.(C) Recombination frequencies in SPD8 cells induced by HU. Cells were treated with 2 mM HU for the times indicated and recovered for 48 hr. HPRT+ revertants were quantified 7 days later. The means and SD (bars) of four to ten independent experiments are shown. Values marked with asterisks are significantly different from control (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; see also Figure S2).
Mentions: To analyze the response of RAD51 and HR at early and late times of replication blocks, we performed time courses of RAD51 foci formation and HR frequencies induced by HU treatment. Cells were treated with HU for different times, and the percentages of cells containing more than 10 RAD51 foci were quantified. RAD51 foci are induced after 24 hr, but not after 1 or 2 hr HU treatment in U2OS cells (Figures 3A and 3B). To analyze the time course of HR induction by HU treatments, we used the SPD8 cell line, which carries a recombination reporter in the hprt gene. HR by either unequal sister chromatid exchange, intrachromatid exchange, single-strand annealing, or gene conversion can lead to restoration of the wild-type hprt gene encoding a functional HGPRT protein (Helleday et al., 1998). We found that HR is induced by HU treatments of 24, but not by treatments of 1 or 2 hr (Figure 3C). This finding suggested that HR is not active while replication forks restart, but is induced when replication forks become inactivated. Because we used SPD8 cells to measure HR, we confirmed that they display similar progressive replication fork inactivation as do U2OS cells (Figures S2A–S2D). Forks become inactivated considerably earlier in SPD8 cells, accompanied by earlier RAD51 foci formation (Figure S2E), which colocalize with stalled replication forks (Figure S2F). DSB induction occurred earlier in SPD8 cells as well, although there was no difference to U2OS cells at 2 hr (Figure S2G). These observations confirm that HR activation occurs late during replication blocks and coincides with or is preceded by replication fork inactivation and DSB formation.

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