Limits...
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.

Show MeSH
XRCC3 Promotes the Early Restart of Stalled Replication Forks(A) Protein levels of XRCC3 and α-Tubulin (loading control) in U2OS cells after 48 hr depletion with control siRNA, pool of 4 XRCC3 siRNAs, or individual XRCC3 siRNAs #1 and #2. The asterisk denotes a nonspecific band.(B) Representative images of replication tracks after restart from 2 hr HU treatment in control- and XRCC3-depleted U2OS cells.(C) Quantification of fork restart in control- or XRCC3-depleted U2OS cells. The means and SD (bars) of at least three independent experiments are shown. Values marked with asterisks are significantly different from control (Student's t test, ∗∗p < 0.01 and ∗∗∗p < 0.001).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2958316&req=5

fig5: XRCC3 Promotes the Early Restart of Stalled Replication Forks(A) Protein levels of XRCC3 and α-Tubulin (loading control) in U2OS cells after 48 hr depletion with control siRNA, pool of 4 XRCC3 siRNAs, or individual XRCC3 siRNAs #1 and #2. The asterisk denotes a nonspecific band.(B) Representative images of replication tracks after restart from 2 hr HU treatment in control- and XRCC3-depleted U2OS cells.(C) Quantification of fork restart in control- or XRCC3-depleted U2OS cells. The means and SD (bars) of at least three independent experiments are shown. Values marked with asterisks are significantly different from control (Student's t test, ∗∗p < 0.01 and ∗∗∗p < 0.001).

Mentions: The XRCC3 protein is required for RAD51 ssDNA complex formation (Bishop et al., 1998). To test whether RAD51 complex formation, which is required for strand invasion (Baumann et al., 1996), is involved in replication restart, we analyzed whether XRCC3 is also required for early restart of replication forks by depleting U2OS cells of XRCC3 using siRNA (Figure 5A). Forty-eight hours later, cells were pulse-labeled with CldU, blocked with HU, and released into IdU for 1 hr. We found that, like RAD51 depletion, XRCC3 depletion increases the number of forks that do not resume replication after release from 2 hr HU block (Figures 5B and 5C). To confirm the specificity of the XRCC3 depletion, we used two individual siRNA duplexes targeting different sequences in the XRCC3 mRNA, with similar results (Figure 5C). These observations demonstrate that, like RAD51, XRCC3 is required to reactivate stalled replication 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)

XRCC3 Promotes the Early Restart of Stalled Replication Forks(A) Protein levels of XRCC3 and α-Tubulin (loading control) in U2OS cells after 48 hr depletion with control siRNA, pool of 4 XRCC3 siRNAs, or individual XRCC3 siRNAs #1 and #2. The asterisk denotes a nonspecific band.(B) Representative images of replication tracks after restart from 2 hr HU treatment in control- and XRCC3-depleted U2OS cells.(C) Quantification of fork restart in control- or XRCC3-depleted U2OS cells. The means and SD (bars) of at least three independent experiments are shown. Values marked with asterisks are significantly different from control (Student's t test, ∗∗p < 0.01 and ∗∗∗p < 0.001).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2958316&req=5

fig5: XRCC3 Promotes the Early Restart of Stalled Replication Forks(A) Protein levels of XRCC3 and α-Tubulin (loading control) in U2OS cells after 48 hr depletion with control siRNA, pool of 4 XRCC3 siRNAs, or individual XRCC3 siRNAs #1 and #2. The asterisk denotes a nonspecific band.(B) Representative images of replication tracks after restart from 2 hr HU treatment in control- and XRCC3-depleted U2OS cells.(C) Quantification of fork restart in control- or XRCC3-depleted U2OS cells. The means and SD (bars) of at least three independent experiments are shown. Values marked with asterisks are significantly different from control (Student's t test, ∗∗p < 0.01 and ∗∗∗p < 0.001).
Mentions: The XRCC3 protein is required for RAD51 ssDNA complex formation (Bishop et al., 1998). To test whether RAD51 complex formation, which is required for strand invasion (Baumann et al., 1996), is involved in replication restart, we analyzed whether XRCC3 is also required for early restart of replication forks by depleting U2OS cells of XRCC3 using siRNA (Figure 5A). Forty-eight hours later, cells were pulse-labeled with CldU, blocked with HU, and released into IdU for 1 hr. We found that, like RAD51 depletion, XRCC3 depletion increases the number of forks that do not resume replication after release from 2 hr HU block (Figures 5B and 5C). To confirm the specificity of the XRCC3 depletion, we used two individual siRNA duplexes targeting different sequences in the XRCC3 mRNA, with similar results (Figure 5C). These observations demonstrate that, like RAD51, XRCC3 is required to reactivate stalled replication 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