brief communications - Nature

Mutations in BLM give rise to Bloom’s syndrome, a genetic disorder associated with cancer predisposition and chromosomal instability. Using a dual-labeling system in isolated chromosome fibers, we show that the BLM protein is required for two aspects of the cellular response to replicative stress: efficient replication-fork restart and suppression of new origin firing. These functions require the helicase activity of BLM and the Thr99 residue targeted by stress-activated kinases. Bloom’s syndrome (BS) is a genetic disorder associated with predisposition to the development of cancer1. The BS-associated gene product BLM is implicated in homologous recombination through the dissolution of double Holliday junctions2. Nevertheless, it is likely that BLM functions primarily during S phase, because BS cells show defects in DNA replication and BLM is recruited to sites of stalled replication forks in cells treated with hydroxyurea (HU)3,4. To analyze replication in more detail in BS cells, we investigated the effects of perturbing DNA synthesis with different classes of

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Sally L Davies, Phillip S North & Ian D Hickson

replication inhibitors. These analyses were conducted using an isogenic pair of cell lines that differ only in their BLM status (designated PSNF5 (BLM+) and PSNG13 (BLM–)). First, we analyzed cell survival. We found that PSNG13 cells were appreciably more sensitive than PSNF5 cells to not only HU (as reported5) but also aphidicolin and the clinically used replication inhibitors gemcytabine and cytosine arabinoside (Supplementary Fig. 1 online). This difference was not due to a significantly greater number of cells being in S phase in PSNG13 than in PSNF5 cultures (44% versus 41%; P 4 0.05). Next, we investigated whether the role of BLM in protecting cells against replicative stress is to facilitate replication-fork restart after blockade of DNA synthesis. We used a dual-labeling protocol (see Fig. 1a and Supplementary Methods online) in which sites of active replication were first marked by incorporation of the nucleoside analog iododeoxyuridine (idU) for a fixed period of time. Replication was then arrested with either HU or aphidicolin. After removing the inhibitor, we assessed the efficiency of replication-fork restart by labeling replicons that were able to resume activity with chlorodeoxyuridine (cldU). Sites of incorporation of idU and cldU were then differentially detected on isolated chromosome fibers using specific antibodies (Fig. 1a). We found that PSNG13 (BLM–) cells showed a progressive loss of replication-fork activity that was more rapid and severe than that in PSNF5 cells (Fig. 1b). The proportion of replication forks that were competent for restarting in the BLM-defective PNSG13 cells declined steadily during exposure to aphidicolin, from


Role for BLM in replication-fork restart and suppression of origin firing after replicative stress


© 2007 Nature Publishing Group http://www.nature.com/nsmb

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Figure 1 BLM is required for efficient replication-fork restart after replication blockade. (a) Images of chromosome fibers with the protocol for defining sites of replication. Where required, a replication inhibitor was added after idU incorporation. Right, diagrammatic representations of possible fiber-labeling patterns. (b) Effects of aphidicolin on replication-fork activity in PSNF5 (BLM+) and PSNG13 (BLM–) cells. (c) Effects of 6-h exposure to HU on replication-fork activity. Error bars, s.e.m. (d) Replication-fork activity in PSNF5 cells (BLM+), PSNG13 cells (BLM–) and cells expressing BLM-K695T. Cells were exposed to no drug, 30 mM aphidicolin for 6 h or 4 mM HU for 6 h, as indicated. Data in b–d are means of three independent determinations; error bars show s.e.m. Cancer Research UK Laboratories, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK. Correspondence should be addressed to I.D.H. ([email protected]). Received 22 March; accepted 6 June; published online 24 June 2007; doi:10.1038/nsmb1267

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Figure 2 The ATR kinase target residue in BLM, Thr99, is required for efficient replication-fork restart and to suppress new origin firing. (a) Replication-fork recovery in the indicated cell lines after a 20-min recovery from exposure to 30 mM aphidicolin for 6 h. (b) Number of new sites of replication visible during a 20-min recovery period after exposure to 30 mM aphidicolin for indicated times. (c) The Thr99 residue in BLM is required to suppress new origin firing after a 6-h exposure to 30 mM aphidicolin or 4 mM HU. (d) Roscovitine (20 mM) suppresses the excessive new origin firing in PSNG13 (BLM–) cells exposed to aphidicolin. Data in a–d are means of three independent determinations; error bars show s.e.m.

80.5% to 45.7% after 6 h of exposure (a 43.2% decline). In contrast, fork recovery in PSNF5 cells declined from 82.5% in the absence of drug to 70.7% after 6 h of aphidicolin treatment (a decline of only 14.3%). One explanation for this difference could be that the kinetics of recovery of DNA synthesis at very early times after replication blockade differs between PSNF5 and PSNG13 cells. To address this possibility, we analyzed DNA synthesis for up to 80 min after the release from a 6-h replication blockade. The result (Supplementary Fig. 2a online) confirmed that BLM-defective PSNG13 cells show defective replication-fork recovery over an extended period. Cell viability during a 6-h exposure to aphidicolin did not fall below 75% for either cell line (data not shown). The fiber analyses were then repeated in cells exposed to HU for 6 h. Consistent with a general replication-restart defect in BS cells, PSNG13 cells were markedly less able to restart replication after HU treatment, compared with PSNF5 cells (Fig. 1c). This defect in replication-fork recovery was also seen in untransformed BS cells (Supplementary Fig. 2b). Hence, BLM is required for efficient replication-fork restart after replication blockade. Previous studies on yeast sgs1 and rqh1 mutants, which have defective BLM orthologs6, have indicated that the DNA helicase activity of RecQ helicases may not be required for all cellular functions. The expression of Sgs1p or Rqh1p with a lysine substitution in the Walker A motif of the ATP-binding site is sufficient to complement some, but not all, defects in sgs1D and rqh1D strains, respectively7–9. We examined whether an equivalent active site substitution in BLM (K695T) affects the ability of BLM to facilitate replication-fork restart. To do this, we analyzed isogenic BS cell lines expressing either no BLM, wild-type BLM or BLM-K695T10. We found that the cells expressing BLM-K695T were as defective as cells lacking BLM in restoring replication-fork activity after exposure to either aphidicolin or HU (Fig. 1d). Consistent with this, the BLMK695T cells were hypersensitive to killing by both of these replication inhibitors (Supplementary Fig. 3a,b online). Together, these results indicate that the ATPase/helicase activity of BLM is required for protection of cells against the cytotoxic effects of DNA synthesis inhibitors and for replication-fork restart after replication blockade. BLM is a target for stress-activated protein kinases after DNA damage or replication blockade5,11. We have shown previously that the ATR kinase phosphorylates Thr99 and Thr122 of BLM in cells treated with HU5. We next investigated whether these phosphorylation

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target residues are important for the function of BLM in replicationfork restart. To do this, we created stable transfectants isogenic to PSNF5 and PSNG13 that express a BLM-T99A or BLM-T122A mutant, or a BLM–T99A T122A double mutant, then analyzed chromosome fibers in these transfectants. Cells expressing BLMT122A behaved similarly to PSNF5 cells in that they maintained robust replication-fork restart activity. In contrast, cells expressing either BLM-T99A or BLM–T99A T122A were as defective in replication restart as cells lacking BLM protein (Fig. 2a). Consistent with these data, BLM-T99A and BLM–T99A T122A cells were hypersensitive to killing by both aphidicolin and HU, whereas BLM-T122A cells were not (Supplementary Fig. 3c,d). We conclude that the Thr99 residue targeted by ATR is essential not only for protection of human cells from the toxic effects of replication inhibitors, but also for the ability of cells to restart DNA synthesis at sites of replication-fork blockade. Trivial explanations for this effect might be that the BLMT99A protein is not expressed at an appropriate level in the transfected cells or that the protein is catalytically compromised. However, we found that expression of BLM-T99A is equivalent to that of wild-type BLM in PSNF5 cells (Supplementary Fig. 4 online) and that purified recombinant BLM proteins with a T99A or T99Q substitution have robust ATPase and helicase activity (data not shown). Next, we analyzed whether BLM-defective cells might show a defect in the suppression of new origin firing during periods of replicative stress. This was undertaken because of the connection discussed above between BLM and ATR, and because visual inspection of fibers isolated from PSNG13 (BLM–) cells revealed an apparent excess of new origin firing compared with that seen in the corrected PSNF5 (BLM+) cells. Hence, we quantified the number of new sites of DNA replication that arose after exposure to aphidicolin. Such sites were defined by their lack of idU incorporation (used to define existing forks) but were labeled by cldU incorporation and therefore appeared as ‘red only’. This section of the study included only those fibers that showed evidence of existing fork activity, to exclude fibers originating from cells that might have entered S phase at the precise time that the aphidicolin or HU was added. PSNF5 cells showed no marked increase in the number of new sites of DNA synthesis after a 6-h exposure to aphidicolin, consistent with the expected ability of cells expressing wild-type BLM to suppress late origin firing (Fig. 2b). In contrast, in PSNG13 cells there was a progressive appearance of new sites of DNA

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B R I E F C O M M U N I C AT I O N S synthesis, indicating that origin firing was not suppressed. After a 6-h treatment with aphidicolin, PSNG13 cells showed an approximately four-fold increase in the number of new sites of DNA synthesis per nanometer of chromosome fiber, compared with PSNF5 cells (Fig. 2b). This defect was seen not only in aphidicolin-treated cells but also in HU-treated cells, and it also required the ATR target residue Thr99 in BLM (Fig. 2c). It is known that the cyclin-dependent kinase inhibitor roscovitine is able to suppress new origin firing while having little or no effect on replication elongation12, and we found that roscovitine suppressed a high proportion of the excessive origin firing seen in PSNG13 cells after exposure to aphidicolin (Fig. 2d). We also tested whether expression of the BLM-K695T active site mutant could suppress excessive new origin firing in BS cells, but it could not (Supplementary Fig. 5 online), indicating that this role of BLM is also dependent upon ATPase/helicase activity. BLM seems to perform a function that couples recombinational repair with the protection of damaged or stalled replication forks. One plausible biochemical role for BLM that would be consistent with the data presented here is to promote replication-fork regression13, a process thought to occur at stalled forks to facilitate replication restart6,14,15. Further work is required to test this hypothesis. We have also identified a role for BLM in suppression of new origin firing that probably indicates cross-talk between replication-fork repair factors and the checkpoint machinery. As this role requires BLM catalysis, we propose that it is dependent upon BLM processing of stalled forks to generate a DNA structure that contributes to checkpoint signaling. Note: Supplementary information is available on the Nature Structural & Molecular Biology website.


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ACKNOWLEDGMENTS We thank members of the CR-UK Genome Integrity Group for helpful discussions, N. Ellis (University of Chicago), and M. Sanz and J. German (Cornell University Medical School, New York) for the wild-type BLM- and BLM-K695T– expressing GM08505 cells, P. McHugh, K. Hanada, C. Bachrati and L. Wu for useful comments on the manuscript, and P. White for preparation of the manuscript. This work was supported by Cancer Research UK. AUTHOR CONTRIBUTIONS Experiments were designed by S.L.D., P.S.N. and I.D.H., and performed by S.L.D. and P.S.N. S.L.D. and I.D.H. interpreted data and wrote the manuscript. COMPETING INTERESTS STATEMENT The authors declare no competing financial interests. Published online at http://www.nature.com/nsmb/ Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions 1. German, J. Medicine 72, 393–406 (1993). 2. Wu, L. & Hickson, I.D. Nature 426, 870–874 (2003). 3. Lonn, U., Lonn, S., Nylen, U., Winbald, G. & German, J. Cancer Res. 50, 3141–3145 (1990). 4. Sengupta, S. et al. EMBO J. 22, 1210–1222 (2003). 5. Davies, S.L., North, P.S., Dart, A., Lakin, N.D. & Hickson, I.D. Mol. Cell. Biol. 24, 1279–1291 (2004). 6. Wu, L. & Hickson, I.D. Annu. Rev. Genet. 40, 279–306 (2006). 7. Ahmad, F. & Stewart, E. Mol. Genet. Genomics 273, 102–114 (2005). 8. Bjergbaek, L., Cobb, J.A., Tsai-Pflugfelder, M. & Gasser, S.M. EMBO J. 24, 405–417 (2005). 9. Lo, Y.C. et al. Mol. Cell. Biol. 26, 4086–4094 (2006). 10. Neff, N.F. et al. Mol. Biol. Cell 10, 665–676 (1999). 11. Beamish, H. et al. J. Biol. Chem. 277, 30515–30523 (2002). 12. Payton, M. et al. Cancer Res. 66, 4299–4308 (2006). 13. Ralf, C., Hickson, I.D. & Wu, L. J. Biol. Chem. 281, 22839–22846 (2006). 14. Cox, M.M. Annu. Rev. Genet. 35, 53–82 (2001). 15. McGlynn, P. & Lloyd, R.G. Nat. Rev. Mol. Cell Biol. 3, 859–870 (2002).

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