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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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Replication Forks Accumulate DNA Damage during Long Replication Blocks(A) Colocalization of HU-induced γH2AX with RPA. Cells were left untreated or treated with 2 mM HU for 2 or 24 hr, fixed, and immunostained for γH2AX (green) and RPA70 (red). DNA was counterstained with DAPI (blue).(B) Quantification of U2OS cells displaying γH2AX immunostaining after 0, 1, 2, or 24 hr of treatment with 2 mM HU and 1 hr after release from 2 or 24 hr HU treatment. Cells containing more than 10 foci were scored as positive. The means and SD (bars) of three independent experiments are shown. Values marked with asterisks are significantly different (∗∗p < 0.01).(C) Pulsed-field gel electrophoresis to visualize DSB induction in U2OS cells treated with 2 mM HU for 2 to 48 hr.(D) Colocalization of γH2AX and stalled replication forks in U2OS cells. Cells were pulse-labeled with CldU for 20 min, treated with 2 mM HU for 2 or 24 hr, and released into IdU for 1 hr. Cells were immunostained for CldU (red), IdU (green), and γH2AX (far red), and DNA was counterstained with DAPI (blue). DNA was denatured with HCl to allow CldU/IdU detection.
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fig2: Replication Forks Accumulate DNA Damage during Long Replication Blocks(A) Colocalization of HU-induced γH2AX with RPA. Cells were left untreated or treated with 2 mM HU for 2 or 24 hr, fixed, and immunostained for γH2AX (green) and RPA70 (red). DNA was counterstained with DAPI (blue).(B) Quantification of U2OS cells displaying γH2AX immunostaining after 0, 1, 2, or 24 hr of treatment with 2 mM HU and 1 hr after release from 2 or 24 hr HU treatment. Cells containing more than 10 foci were scored as positive. The means and SD (bars) of three independent experiments are shown. Values marked with asterisks are significantly different (∗∗p < 0.01).(C) Pulsed-field gel electrophoresis to visualize DSB induction in U2OS cells treated with 2 mM HU for 2 to 48 hr.(D) Colocalization of γH2AX and stalled replication forks in U2OS cells. Cells were pulse-labeled with CldU for 20 min, treated with 2 mM HU for 2 or 24 hr, and released into IdU for 1 hr. Cells were immunostained for CldU (red), IdU (green), and γH2AX (far red), and DNA was counterstained with DAPI (blue). DNA was denatured with HCl to allow CldU/IdU detection.

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.


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)

Replication Forks Accumulate DNA Damage during Long Replication Blocks(A) Colocalization of HU-induced γH2AX with RPA. Cells were left untreated or treated with 2 mM HU for 2 or 24 hr, fixed, and immunostained for γH2AX (green) and RPA70 (red). DNA was counterstained with DAPI (blue).(B) Quantification of U2OS cells displaying γH2AX immunostaining after 0, 1, 2, or 24 hr of treatment with 2 mM HU and 1 hr after release from 2 or 24 hr HU treatment. Cells containing more than 10 foci were scored as positive. The means and SD (bars) of three independent experiments are shown. Values marked with asterisks are significantly different (∗∗p < 0.01).(C) Pulsed-field gel electrophoresis to visualize DSB induction in U2OS cells treated with 2 mM HU for 2 to 48 hr.(D) Colocalization of γH2AX and stalled replication forks in U2OS cells. Cells were pulse-labeled with CldU for 20 min, treated with 2 mM HU for 2 or 24 hr, and released into IdU for 1 hr. Cells were immunostained for CldU (red), IdU (green), and γH2AX (far red), and DNA was counterstained with DAPI (blue). DNA was denatured with HCl to allow CldU/IdU detection.
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fig2: Replication Forks Accumulate DNA Damage during Long Replication Blocks(A) Colocalization of HU-induced γH2AX with RPA. Cells were left untreated or treated with 2 mM HU for 2 or 24 hr, fixed, and immunostained for γH2AX (green) and RPA70 (red). DNA was counterstained with DAPI (blue).(B) Quantification of U2OS cells displaying γH2AX immunostaining after 0, 1, 2, or 24 hr of treatment with 2 mM HU and 1 hr after release from 2 or 24 hr HU treatment. Cells containing more than 10 foci were scored as positive. The means and SD (bars) of three independent experiments are shown. Values marked with asterisks are significantly different (∗∗p < 0.01).(C) Pulsed-field gel electrophoresis to visualize DSB induction in U2OS cells treated with 2 mM HU for 2 to 48 hr.(D) Colocalization of γH2AX and stalled replication forks in U2OS cells. Cells were pulse-labeled with CldU for 20 min, treated with 2 mM HU for 2 or 24 hr, and released into IdU for 1 hr. Cells were immunostained for CldU (red), IdU (green), and γH2AX (far red), and DNA was counterstained with DAPI (blue). DNA was denatured with HCl to allow CldU/IdU detection.
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.

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