Materials and Methods Cell lines and cell culture. GM08505 is an SV40-transformed skin fibroblast cell line derived from a BS patient and contains the BLM Ash founder mutation identified in patients of Ashkenazi Jewish origin 1. The PSNF5 and PSNG13 cells have been described previously 2,3 and were derived from GM08505 cells following transfection with a construct expressing flag epitope-tagged BLM protein or the pcdna3 vector, respectively. PSNF5 cells are considered to be phenotypically corrected in that the high frequency of sister-chromatid exchanges (SCEs) diagnostic of BS cells is suppressed to near normal levels in this cell line 2,3. Stable transfectants of GM08505 cell line expressing BLM-T99A, BLM-T122A or BLM- T99A/T122A have been described previously 2. The GM08505 derivatives expressing wildtype BLM or BLM-K695T protein were a kind gift of M. Sanz and J. German, and were described elsewhere 4. All cells were grown in α-mem (minimal essential medium) culture medium containing 10% fetal calf serum and 3mM glutamine at 37 C in a humidified atmosphere containing 5% CO 2. DNA replication inhibitors and clonogenic cell survival analyses. Aphidicolin, HU, gemcytabine and cytosine arabinoside (Ara C) were obtained from Sigma. The CDK inhibitor, roscovitine, was obtained from Calbiochem. Aphidicolin was dissolved in DMSO, and the other agents were dissolved in water. Clonogenic survival analyses were performed as described by Davies et al. 2.
Western blotting. Cell extracts were prepared and Western blotting for BLM was conducted using the IHIC34 rabbit polyclonal antibody, as described by Wu et al. 5. β-tubulin was used as a loading control and was detected using the tub2.1 monoclonal antibody (Sigma). Preparation and immunolabeling of chromosome fibers. Cells were exposed to 20 M IdU for 10 minutes to label sites of active replication. Following a 15 minute exposure to 50 M thymidine, cells were left untreated or were exposed to either aphidicolin (30 M) or HU (4mM) for up to 6 hours. The cells were then incubated in drug-free medium for 20 minutes to allow for replication fork re-start in the presence of 100 M CldU, unless stated otherwise. Chromosome spreads were prepared as described by Jackson and Pombo 6 with the modifications described by Merrick et al. 7. Slides were acid treated with 2.5M HCl for 1 hour, neutralized in 0.1M Na 3 B 4 O 7, ph 8.5, for 7 minutes, and were then washed several times in phosphate buffered saline (PBS). Samples were then blocked for 30 minutes with 1% BSA and 0.1% Tween 20 in PBS. Antibodies were diluted in blocking buffer as follows: anti-cldu, 1:40; anti-mouse Alexafluor 488, 1:200; anti-idu, 1:2; anti-rat Cy3, 1:900. Incubations with antibodies were carried out at 37 C for 1 hour (for primary antibodies) or 45 minutes (for secondary antibodies). The incubation with the IdU antibody was followed by a high salt wash with buffer (29.2g NaCl, 4.44g Tris-HCI, 0.5% Tween 20 and 1 litre H 2 O) at 20 C for 7 minutes, to increase antibody specificity and discrimination. Slides were mounted in anti-fade (90% glycerol, 20mM Tris-HCl, ph 8.0, 50 g/ml paraphenylenediamine) prior to analysis using a Zeiss Axioskop microscope. Replication fork activity was calculated by dividing the number of sites of continuing replication [A+B; as depicted on the right of panel (a)] by the total number of IdU-containing sites [A+B+D]. More than 50 individual fibers were analyzed in each experiment and the data presented represent the mean of at least 3 independent experiments.
Supplementary References 1. Ellis, N.A. et al. Cell 83, 655-666 (1995). 2. Davies, S.L., North, P.S., Dart, A., Lakin, N.D. & Hickson, I.D. Mol. Cell. Biol. 24, 1279-1291 (2004). 3. Gaymes, T.J. et al. Oncogene 21, 2525-2533 (2002). 4. Neff, N.F. et al.. J. Biol. Chem. 275, 9636-9644 (2000). 6. Jackson, D.A. & Pombo, A. J. Cell Biol. 140, 1285-95 (1998). 7. Merrick, C.J., Jackson, D. & Diffley, J.F. J. Biol. Chem. 279, 20067-75 (2004).
Supplementary Figure Legends Supplementary Figure 1. BS cells are hypersensitive to drugs that inhibit DNA replication. Clonogenic cell survival analyses were conducted on PSNF5 (BLM + ; ) and PSNG13 (BLM - ; ϒ) cells following a 24 hour exposure to aphidicolin (panel a), gemcytabine (panel b) or ara C (panel c) at the doses indicated. Experiments were performed in triplicate and the error bars represent the standard errors of the mean. Supplementary Figure 2. BLM is required for efficient replication fork restart following replication blockade. (a) Effect of varying recovery time on replication fork activity following aphidicolin treatment of 30 M for 6 hours. (b) Effects of a 6 hour exposure to aphidicolin or HU compared to untreated controls in untransformed MRC5 cells (BLM + ; black bars) and GM1492 (BLM - ; open bars) cells. Supplementary Figure 3. The active site lysine (K695) and the Thr-99 target site for ATR are required for cell survival after replication blockade. Survival curves were generated for the cell lines indicated as described in the Supplementary Figure 1 legend. Panels (a) and (c) show sensitivity to aphidicoln and panels (b) and (d) show sensitivity to HU. Supplementary Figure 4. Expression of BLM proteins in transfected BS cells. Western blotting analysis to indicate levels of the BLM, BLM-T99A, BLMT122A, and BLM- T99A/T122A proteins in the different transfectants. β-tubulin was used as a loading control. Supplementary Figure 5. The active site of BLM is required for suppression of new origin firing. Number of new sites of replication visible during a 20 minute recovery period
following exposure to 30 M or 4mM HU in an isogenic set of cell lines expressing wild-type BLM (black), no BLM (white) or BLM-K695T (red).