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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 Restart after Short Replication Blocks but Become Inactivated during Long Replication Blocks(A) Labeling protocols for DNA fiber and replication foci analysis. U2OS cells were pulse-labeled with CldU, treated with 2 mM HU for the times indicated, and released into IdU. CldU was detected using a specific primary antibody and a secondary antibody in red. IdU was detected using specific primary antibody and a secondary antibody in green.(B) Representative images of replication tracks from U2OS cells after release from 1, 2, or 24 hr HU treatment.(C) Quantification of fork restart in U2OS cells after release from 1, 2, or 24 hr HU treatment.(D) Global replication restart in U2OS cells after release from 2 or 24 hr HU treatment. Cells were pulse-labeled as in (A), then fixed and immunostained for CldU (red) and IdU (green). DNA was counterstained with DAPI (blue).(E) Quantification of cells restarting global replication as in (D). Cells labeled with both CldU and IdU are shown as percentages of all cells labeled with CldU.(F) Quantification of cells newly entering S phase after release from 2 or 24 hr HU treatment. Cells labeled with only IdU are shown as percentages of total cells. The means and standard deviation (SD) (bars) of at least three independent experiments are shown. Values marked with asterisks are significantly different (Student's t test, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; see also Figure S1).
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fig1: Replication Forks Restart after Short Replication Blocks but Become Inactivated during Long Replication Blocks(A) Labeling protocols for DNA fiber and replication foci analysis. U2OS cells were pulse-labeled with CldU, treated with 2 mM HU for the times indicated, and released into IdU. CldU was detected using a specific primary antibody and a secondary antibody in red. IdU was detected using specific primary antibody and a secondary antibody in green.(B) Representative images of replication tracks from U2OS cells after release from 1, 2, or 24 hr HU treatment.(C) Quantification of fork restart in U2OS cells after release from 1, 2, or 24 hr HU treatment.(D) Global replication restart in U2OS cells after release from 2 or 24 hr HU treatment. Cells were pulse-labeled as in (A), then fixed and immunostained for CldU (red) and IdU (green). DNA was counterstained with DAPI (blue).(E) Quantification of cells restarting global replication as in (D). Cells labeled with both CldU and IdU are shown as percentages of all cells labeled with CldU.(F) Quantification of cells newly entering S phase after release from 2 or 24 hr HU treatment. Cells labeled with only IdU are shown as percentages of total cells. The means and standard deviation (SD) (bars) of at least three independent experiments are shown. Values marked with asterisks are significantly different (Student's t test, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; see also Figure S1).

Mentions: To understand the fate of replication forks following replication blocks, we analyzed the restart of replication forks after different periods of HU treatment using the DNA fiber technique (Henry-Mowatt et al., 2003). HU depletes deoxyribonucleotide pools and immediately stalls replication forks (Bianchi et al., 1986). U2OS cells were pulse-labeled with 5-chlorodeoxyuridine (CldU) for 20 min, washed and blocked in HU for 2 hr, washed again and pulse-labeled with 5-iododeoxyuridine (IdU) for 1, 2, or 24 hr (Figure 1A). Afterward, DNA spreads were prepared and analyzed by immunofluorescence (Figure 1B). To quantify replication fork restart, the amount of stalled forks was related to the total number of replication tracks labeled with CldU (Figure 1C). Surprisingly, we found that, although most forks restarted after release from a 1 or 2 hr HU block, most forks remained stalled after release from 24 hr HU block. Instead of restarting forks, replication tracks labeled only with IdU appeared that seemed to result from new initiation events (Figures 1B and 1C). Because it was previously shown that nucleotide incorporation resumes between 12 and 18 hr of HU blocks (Hanada et al., 2007), we included IdU during the HU treatment to determine whether the apparent lack of restart was due to forks moving large distances during the treatment, resulting in the two labels becoming separated (see Figure S1A available online). Most forks moved less than 6 μm (15.5 kb) during the 24-hr block (Figures S1B and S1C). CldU tracks in less than 6 μm distance from IdU tracks were therefore not considered as stalled forks. Taking this into consideration, our data suggest that most forks become inactivated after long times in HU.


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 Restart after Short Replication Blocks but Become Inactivated during Long Replication Blocks(A) Labeling protocols for DNA fiber and replication foci analysis. U2OS cells were pulse-labeled with CldU, treated with 2 mM HU for the times indicated, and released into IdU. CldU was detected using a specific primary antibody and a secondary antibody in red. IdU was detected using specific primary antibody and a secondary antibody in green.(B) Representative images of replication tracks from U2OS cells after release from 1, 2, or 24 hr HU treatment.(C) Quantification of fork restart in U2OS cells after release from 1, 2, or 24 hr HU treatment.(D) Global replication restart in U2OS cells after release from 2 or 24 hr HU treatment. Cells were pulse-labeled as in (A), then fixed and immunostained for CldU (red) and IdU (green). DNA was counterstained with DAPI (blue).(E) Quantification of cells restarting global replication as in (D). Cells labeled with both CldU and IdU are shown as percentages of all cells labeled with CldU.(F) Quantification of cells newly entering S phase after release from 2 or 24 hr HU treatment. Cells labeled with only IdU are shown as percentages of total cells. The means and standard deviation (SD) (bars) of at least three independent experiments are shown. Values marked with asterisks are significantly different (Student's t test, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; see also Figure S1).
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fig1: Replication Forks Restart after Short Replication Blocks but Become Inactivated during Long Replication Blocks(A) Labeling protocols for DNA fiber and replication foci analysis. U2OS cells were pulse-labeled with CldU, treated with 2 mM HU for the times indicated, and released into IdU. CldU was detected using a specific primary antibody and a secondary antibody in red. IdU was detected using specific primary antibody and a secondary antibody in green.(B) Representative images of replication tracks from U2OS cells after release from 1, 2, or 24 hr HU treatment.(C) Quantification of fork restart in U2OS cells after release from 1, 2, or 24 hr HU treatment.(D) Global replication restart in U2OS cells after release from 2 or 24 hr HU treatment. Cells were pulse-labeled as in (A), then fixed and immunostained for CldU (red) and IdU (green). DNA was counterstained with DAPI (blue).(E) Quantification of cells restarting global replication as in (D). Cells labeled with both CldU and IdU are shown as percentages of all cells labeled with CldU.(F) Quantification of cells newly entering S phase after release from 2 or 24 hr HU treatment. Cells labeled with only IdU are shown as percentages of total cells. The means and standard deviation (SD) (bars) of at least three independent experiments are shown. Values marked with asterisks are significantly different (Student's t test, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; see also Figure S1).
Mentions: To understand the fate of replication forks following replication blocks, we analyzed the restart of replication forks after different periods of HU treatment using the DNA fiber technique (Henry-Mowatt et al., 2003). HU depletes deoxyribonucleotide pools and immediately stalls replication forks (Bianchi et al., 1986). U2OS cells were pulse-labeled with 5-chlorodeoxyuridine (CldU) for 20 min, washed and blocked in HU for 2 hr, washed again and pulse-labeled with 5-iododeoxyuridine (IdU) for 1, 2, or 24 hr (Figure 1A). Afterward, DNA spreads were prepared and analyzed by immunofluorescence (Figure 1B). To quantify replication fork restart, the amount of stalled forks was related to the total number of replication tracks labeled with CldU (Figure 1C). Surprisingly, we found that, although most forks restarted after release from a 1 or 2 hr HU block, most forks remained stalled after release from 24 hr HU block. Instead of restarting forks, replication tracks labeled only with IdU appeared that seemed to result from new initiation events (Figures 1B and 1C). Because it was previously shown that nucleotide incorporation resumes between 12 and 18 hr of HU blocks (Hanada et al., 2007), we included IdU during the HU treatment to determine whether the apparent lack of restart was due to forks moving large distances during the treatment, resulting in the two labels becoming separated (see Figure S1A available online). Most forks moved less than 6 μm (15.5 kb) during the 24-hr block (Figures S1B and S1C). CldU tracks in less than 6 μm distance from IdU tracks were therefore not considered as stalled forks. Taking this into consideration, our data suggest that most forks become inactivated after long times in HU.

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