SUPPLEMENTARY INFORMATION. doi: /nature Human 1 Mouse 1 Xenopus Human 2 Mouse 2 Danio Dros Sulso

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1 S1 Human 1 Mouse 1 Xenopus Human 2 Mouse 2 Danio Dros Sulso Human 1 Mouse 1 Xenopus Human 2 Mouse 2 Danio Dros Sulso Human 1 Mouse 1 Xenopus Human 2 Mouse 2 Danio Dros Sulso Human 1 Mouse 1 Xenopus Human 2 Mouse 2 Danio Dros Sulso Figure S1. Database mining revealed two human SSB homologues. Alignments of eukaryotic (human, mouse, Xenopus laevis, Danio rerio, Drosophila melanogaster) SSBs and an archaeal (Sulfolob us solfataricus) SSB showing a highly conserved N- terminal OB fold domain followed by a variable region with no predicted structure and a conserved C-terminal tail. 1

2 S2 Figure S2. specifically interacts with ssdna. The upper panel shows an electrophoretic mobility shift experiment with increasing concentrations of (0, 2, 3, 6, 10!M) incubated with d30t DNA (70 fmol) in gel shift buffer (10 mm Tris ph 7.5, 50 mm NaCl, 1 mm MgCl 2, 0.5 mm EDTA, 0.5 mm DTT, 4% glycerol and 1!g/mL BSA) for 30 min at 37 C. Samples were electrophoresed through a 10% native polyacrylamide gel and visualised by phosphorimaging. The lower panel shows the same gel, immunoblotted with a antibody. is present in the bandshifted protein:dna complex. S3 1 Fraction DNA bound d C dac dgt dt da Oligonucleotide d30c d30t d30gt d30ac d30a Apparent Kd (!M) No binding dimer [µm] (µm) Figure S3. Data from triplicate gel shift experiments showing binding curves of using different oligonucleotides. The fraction of DNA bound is plotted against the protein concentration. preferentially binds pyrimidines with no binding to poly A sequences and a lower binding observed if the oligo alternates with dac. The apparent Kds are represented in the table. 2

3 S4 a) d21t d30t d60t Kd ~446nM Kd ~382nM Kd ~193nM b) [] [] [] [] d30t d40t d50t d60t Figure S4. Binding affinity of with longer oligonucleotides. a, Isothermal Titration Calorimetry (ITC) study of the binding of to poly-dt DNA of varying lengths in 20 mm HEPES ph 7.3, 100 mm KCl, 1mM MgCl 2. Oligonucleotide (40!M) was titrated into a solution of (4!M). Raw and integrated binding isotherms are shown in the top panel (inset shows blank titration of buffer into protein). Titrations are in triplicate ± standard deviation. Binding is exothermic, as seen for other OB-fold containing SSB proteins. The binding affinity increases with increasing DNA length. The Kd is indicated at the bottom each panel. b, These data can also be reproduced in an EMSA with the indicated DNA oligonucleotides. The trend is the same although binding is weaker due to the nature of the gelshift experiment and the likely fast on/off rates of the :DNA interaction. 3

4 S5 a) hssb 1 b) c) 1 2 d) kda sicontrol si hssb2 Figure S5. Purification, sirna mediated reduction and antibody characterisation of. a, Purification of recombinant. Recombinant His-tagged was expressed in E.coli and purified initially by nickle-affinity chromatography followed by heparin column and size exclusion chromatography. b, s irna m ediated reduction of protein expression. Cells were transfected twice at 24h intervals with control or sirna and harvested 48h later. Cell extracts were immunoblotted with anti or anti- antibodies as a control for equal protein loading. The antibody specifically recognises a band of 35kDa in cells treated with control s irna and this band is significantly diminished in cells transfected with sirna. c, Anti antibodies do not recognise hssb2. Immunoblot of purified recombinant (lane 1) and hssb2 (lane 2). d, Coomassie-stained protein gel showing levels and migration of purified and hssb2. S6 MG IR NS Figure S6. is stabilised following MG132 treatment. Immunoblot analysis of using cell extracts from neonatal foreskin fibroblasts (NFF) cells exposed to IR (6 Gy) and/or proteosome inhibitor (MG132 for 2 h). Cells were harvested and immunoblotted for. S7 DMSO Wortmannin AT cells vector+atm vector Figure S7. IR-induced stabilisation is ATM-dependent. Mock treated or irradiated (6Gy) cell extracts from NFF cells pre-treated for 1h with wortmannin (20!M) or from ATM-deficient cell lines containing either vector alone or ATM cdna were immunoblotted with the indicated antibodies. 4

5 S8 IP: NS Cell line - L3 C3ABR L3 C3ABR ATM IP: NS ATM Cell line C3ABR ATM Figure S8. In vivo association of and ATM. Cells were irradiated with 6 Gy, incubated for 1 h and lysates were immunoprecipitated using anti- or anti-atm antibodies or pre-immune serum (NS). Associations were detected via immunoblotting with either anti-atm or anti- antibodies. S Amino acid GST-ATM GST-ATM Figure S9. interacts with GST-ATM fragment 4 (amino acids ). Recombinant GST-ATM fragments representing the full length of ATM were used in pulldown assays 4. Total cell extracts from unirradiated (-) and irradiated (+) cells were mixed with glutathione agarose beads containing GST-ATM fusion proteins. Bound proteins were analysed by immunoblotting with anti- antibodies (bottom panel) and levels of GST-ATM fragments were detected by coomassie staining (top panel). 5

6 S10 a) T117A b) hssb T 117A T117E MG GFP GFP Figure S10. IR-induced stabilisation of wild type (GFP-) and phospho-mutant (GFP-T117A). a, Cells were transfected with the indicated constructs and mock treated or irradiated (6Gy). Cell extracts were taken and immunoblotted with anti-gfp antibodies. b, MG132-induced stabilisation of wild-type (GFP-), T117A and T117E mutants. Cells were treated with MG132 for 2h and processed for western blotting with anti-gfp antibodies. S11 a) 1 b) Molar Ellipticity (deg cm 2 /dmol) T117A T117E T117A nm Figure S11. Structural and activity analysis of wild type and mutants (T117A and E). a, The circular dichroism (CD) spectra of the purified recombinant proteins were measured in the far UV range ( nm) in 10 mm Phosphate ph 7.6 and 150 mm NaF at 20 C. A negative mean residue ellipticity (~ -3 mdegree.cm 2.dmol -1 ) was observed at 202 nm. Secondary structure of wild-type and mutants was assessed using the CDPro software package, which includes three analysis programs (CONTIN, CDSSTR, and SELCON3) and show similar secondary structural properties, ~30%!- sheet (consistent with the presence of an OB fold domain (! barrel) in the protein sequence making up approximately of the 245 residues), ~10%, alpha helix and ~25% of the protein estimated as turns. b, Electrophoretic mobility shift assay (EMSA) showing binding of recombinant wild type and phospho-mutant (T117A) to ssdna substrate (d30t). The amounts of used in the EMSA assay were 0, 0.5, 1, 2.5, 5 µm. 6

7 S12 RPA34!H2AX merge enlarged Olympus BX61 IR Deltavision Personal DV IR Figure S12. Comparison of RPA34 and "H2AX colocalisation on Olympus BX61 and Deltavision personal DV microscopes. Cells were treated with 6Gy IR, fixed 1h after treatment, stained with the indicated antibodies and examined via immunoflurescence. On a standard wild-field microscope (Olympus BX61) RPA and "H2AX appear to colocalise after IR, consistent with other reports. In contrast, high resolution images acquired using the Deltavision microscope show that the foci are proximal, and not coincident. Under our experimental conditions RPA and "H2AX do colocalise after hydroxyurea treatment (data not shown), but not after IR treatment, confirming that they do colocalise under certain condtions. S13 0 hours 8 hours 16 hours sicontrol BrdU content si DNA content Figure S13. Normal S-phase progression in -deficient cells. Cells were transfected with control or sirna, pulse-labeled with BrdU and harvested at the indicated time-intervals. The cells were stained with anti-brdu antibodies and PI and analysed by FACS to measure DNA synthesis (BrdU-Alexa 488) and DNA content (PI). Like control sirna transfected cells, virtually all deficient S-phase cells move to G2 (acquire 4N DNA content) by 8h after labelling with BrdU. The boxes indicate the S-phase population. 7

8 S14 DNA content Figure S14. Defective G2/M checkpoint in -deficient cells. NFF cells were treated as indicated, harvested 1 h later and stained with phospho-histone H3 antibodies. The percentage of mitotic cells (boxed area) were determined via FACS. S15 sicontrol si Cdc25A Figure S15. Lack of Cdc25A degradation in sirna transfected cells. NFF cells were transfected with the indicated sirna, irradiated (6Gy) and protein was extracted 1h later. Cell lysates were immunoblotted with the indicated antibodies. S16 si sicontrol p53 Ser15 " -H2AX NS Figure S16. is required for IR-induced ATM-mediated phosphorylation events. A second set of sirna oligos targeted to a different region in, were transfected into NFF cells. Cells were irradiated (6 Gy) and harvested after 1h for western blot analysis with the indicated antibodies. 8

9 S control sirna control sirna A-T Survival (%) (%) 10 sicontrol si 1 # Absorbed dose (Gy) IR dose [Gy] Fig S17. Clonogenic survival of cells with ~50% reduction in expression of. Cells were transfected once with the relevant sirna and irradiated with indicated dose of radiation. Clonogenic survival was determined 13 days following irradiation. Values shown represent the mean results of 3 independent experiments. The A-T cells used are htert-immortalised GM5823 fibroblasts. S18 sicontrol si Figure S18. Metaphase spreads showing severe spontaneous chromatin fragmentation in ~10% of -depleted cells. Fluorescent in situ hybridization (FISH) of metaphase spreads with a telomere-specific PNA probe (red) and centromere-specific PNA probes (green) as described recently

10 S19 Relative I-SceI-induced HRR I-SceI + I-SceI 0 sicon. sicont. si si sicontrol si Figure S19. HR repair is reduced in cells transfected with sirna. 24 h after sirna transfection MCF7-DRGFP cells were transfected with I-Sce1 plasmid (pcbsce). Forty-eight hours after pcbsce transfection FACS analysis was carried out to detect GFP positive cells. S20 a) b) Rad51 Rad51+DNA c) sicontrol si sicontrol Rad51 NBS1 si Figure S20. regulates Rad51 foci formation. a, SiRNA transfected NFF cells were treated with 6Gy IR and cell extracts immunoblotted with the indicated antibodies. b, Rad51 foci formation after IR exposure. NFF cells were transfected with control and sirna and irradiated with 6 Gy IR. Cells were pre-extracted with triton-x-100 before fixation and stained with Rad51 antibodies (Merck). Images were acquired on an Olympus BX61 microscope. c, The percentage of control and -deficient cells showing Rad51 foci after IR. The histogram bars correspond to the average percentage of Rad51 foci positive cells from three independent experiments. 10

11 S21 Rad51 + Rad51 merge Figure S21. Colocalisation of and Rad51. NFF cells were irradiated and 2.5 h later fixed and stained with anti- and anti-rad51 antibodies. Images were acquired using Deltavision Personal DV. Enlargements of the images are displayed in the lower panels demonstrating proximity and colocalisation (yellow) of and Rad51 foci. S22 NS anti- Rad51 Figure S22. In vivo association of and Rad51. Cells were irradiated with 6 Gy, incubated for 2.5 h and lysates were immunoprecipitated using anti- antibodies or pre-immune serum (NS). Associations were detected via immunoblotting with either anti-rad51 or anti- antibodies. 11