SUPPLEMENTARY MATERIALS AND METHODS

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1 SUPPLEMENTARY MATERIALS AND METHODS Chemicals. All chemicals used in supplementary experiments were the same as in the manuscript except the following. Methyl-methanesulfonate (MMS) was from Fluka. NU1025 (Calbiochem) was dissolved in DMSO (10 mm stock solution) and stored at -20 C. Small aliquots of stock solutions chemicals were used once. Antibodies. All antibodies used in supplementary experiments were the same as in the manuscript except the following. Monoclonal antibody anti-xrcc1 Ab-1 (33-2-5) and polyclonal antibody anti- PARP Ab-2 (RB-1516-p1) were from Neomarkers. Anti-DNA ligase III monoclonal antibody was from Transducion Laboratories. DNA-damaging treatments. For MMS treatment, exponentially growing cells were seeded one night before treatment, washed and either mock-treated or treated with freshly diluted MMS in 50, 100, and 200 μg/ml with serum-free DMEM at 37 C for 30 min. For Cali treatment, the procedure was the same as in the manuscript. When indicated, the cells wre pre-treated with freshly diluted specific PARP inhibitor - NU1025 in 200 nm with serum-free DMEM for 30 minutes before MMS treatment. Cells were then harvested and subjected to biochemical fractionation and immuno-blotting. Biochemical fractionation. All the procedures performed in supplementary experiments were similar to those in manuscript but with minor modifications. The incubation time for the first fractionation step (fraction P1) was prolonged to 40 minutes instead of 10 minutes. After pelleted and treated with RNaseA (see materials and methods in manuscript), the pellet (fraction P2) was washed with extraction buffer without Triton, resuspended in 100μl of the same buffer and sonicated for 10 seconds (Vibracel, Bioblock Scientific). Sonicated samples were than supplemented with concentrated sample buffer to final 1 fold (50 mm Tris.HCl ph 6.8, 10% glycerol, 1% SDS, 300 mm 2-mercaptoethanol, 0.01% bromophenol blue) and analysed by immuno-blotting as described in manuscript. LEGENDS TO SUPPLEMENTARY FIGURES Supplementary Figure 1 - Comparison of the recruitment of NHEJ and BER proteins in P2 fraction of MRC5-SV2 cells after treatment with Cali or MMS. Nu1025 is a potent PARP inhibitor (see supplemental Material and methods). Supplementary Figure 2 - Effect of defect on the NHEJ proteins mobilization in response to DNA DSBs. BuS and BuC cells in culture dishes were treated or not with Cali for one hour at 37 C. Cells were collected and lysed in denaturating buffer (WCE) or fractionated by two consecutive extractions as described in the material and methods section, leading to S1 soluble fraction and P2 insoluble material.

2 Supplementary Figure 3 - Consequences of various NHEJ defects on mobilization in response to DNA DSBs in HeLa clones. Various exposures of the upper western blot of Figure 5B with anti and anti antibodies. Supplementary Figure 4 - Consequences of a ligase IV defect on mobilization in response to DNA DSBs in MRC5. Western blotting on WCE and the P2 fraction of the MRC5-SV control and lig4sh clones after treatment with 10 nm Cali for one hour at 37 C. Protein samples were denatured and separated on SDS-PAGE gels 10% for standard separation or 15 % for H2AX isolation. Supplementary Figure 5 - Immunoprecipitation controls. A/ Whole cell protein extracts (WCE) from BuS and BuC cells obtained as described in the material and methods section were diluted in IP buffer and incubated with immunobeads coated with anti-xlf primary antibodies. Then, the extracts supernatant was removed and the beads were washed. Proteins in the immunoprecipitates were heated in SDS sample buffer and separated in 10% SDS-PAGE. Western blotting was performed with antibodies as indicated. B/ Protein extracts from Nalm6 (lig4 + ) and N114P2 (Lig4 - ) cells were incubated with immunobeads coated with IgG from a rabbit control serum and then processed as in Figure 6.

3 Extraction : WCE P2 Cali : + NU1025 : + MMS : PARP XRCC1 Ligase III -actin -H2AX Supplementary Figure 1

4 Extraction: WCE P2 S1 Cells : BuC/BuS BuC BuS BuC BuS Ligase IV Ku80 actin Cali : H2AX Supplementary Figure 2

5 Extraction : P2 Hela clone : ctrl. shl4 shx4 shpkcs Cali : X1 X2 X C1 C2 C3 -actin Supplementary Figure 3

6 Extraction : WCE P2 MRC5 clone : shl4 Vec shl4 Vec Ligase4 Ku80 Ku70 Cali : + + actin Supplementary Figure 4

7 A WCE IP: XLF Cells: BUS BUC BUS BUC B IP: IgG WCE Cells: Nalm6 N114p2 Nalm6 N114p2 Supplementary Figure 5