<p>Supplementary Information</p><p>Materials and Methods</p><p>Chemicals, reagents and antibodies </p><p>Okadaic acid sodium salt, Phosphatase Inhibitor Cocktail 2, wortmannin, Phosphatase </p><p>Inhibitor Cocktail 1 and 4-amino-1, 8-naphthalimide (4ANI) and Fostriecin sodium salt were from Sigma-Aldrich (St-Louis, MO). Tautomycetin was from Calbiochem. Calf </p><p>Intestinal Alkaline Phosphatase (CIP) was from New England Biolabs Inc. The anti-</p><p>PARP-1 (H-250) antibody, the anti-Ku-86 (B-1) antibody and the anti-WRN (H-300) antibody, purchased from Santa Cruz Biotechnology (Santa Cruz, CA ) were used for western blot analysis. For immunoprecipitation an anti-PARP-1 polyclonal antibody from</p><p>Cell Signaling Technology (Danvers, MA) was employed. The anti-Ku (p70/p80) Ab-3 </p><p>(clone 162) antibody, used for immunoprecipitation, was purchased from Lab Vision </p><p>Corporation (Fremont, CA). The anti-β-actin antibody (clone AC-15) was obtained from </p><p>Sigma-Aldrich (St-Louis, MO). The anti-phospho-histone H2AX (ser139) and the anti- nucleolin antibodies were from Upstate Cell Signaling Solutions (Lake Placid, NY). The anti-Poly(ADP-ribose) (10H) antibody was obtained from Alexis Biochemicals (San </p><p>Diego, CA). The anti-PP5/PPT monoclonal antibody was from BD Biosciences </p><p>(Mississauga, ON). PP5 cDNA, amplified from a HeLa cDNA library, was in pFLAG-</p><p>CMV-2 (Sigma-Aldrich) with an N-terminal FLAG tag. Cell culture and treatments </p><p>HeLa, V15B, V15B + Ku80 (Schild-Poulter et al., 2003), V79 cells and M059J derivatives were maintained in DMEM containing 10% serum. HCT116 cells were grown as monolayers in McCoy's 5A medium containing 10% serum. γ-ray irradiation was performed by exposing cells to 137Cs γ-rays from Gammacell 3000 Elan (MDS Nordion, </p><p>Canada) at a dose rate of 5 Gy/min. UV irradiation (25 J/m2) was performed by exposing cells to germicidal UV lamps for 30 sec. Cells were allowed to recover for 0-2 h prior to lysis. </p><p>For siRNA suppression, HeLa cells were transfected with 200 pmol of annealed control double-stranded RNA (acuaccguuguuauaggug) or 100 pmol of ON-TATGETplus </p><p>SMARTpool siRNA targeting PP5C (Dharmacon, Chicago, IL) using Oligofectamine </p><p>Reagent (Invitrogen, Carlsbad, CA ). Cell extracts were prepared 3 days after the transfection. Overexpression of PP5 was carried out in HCT116 cells transfected with pFLAG-CMV-2 or pFLAG-CMV-2 PP5 using FuGene 6 transfection Reagent (Roche) for 3 days prior to treatment and protein extraction. </p><p>For in vivo [32P]-orthophosphate labeling, cells were incubated in phosphate- and serum- free DMEM medium for 2 hours and then labeled for 4 hours with [32P]-orthophosphate </p><p>(200 µCi/mL). </p><p>Immunofluorescence analysis of poly (ADP-ribose) (PAR)</p><p>Hela cells and murine embryonic fibroblasts were fixed in 4% paraformaldehyde in PBS for 10 min, rinsed twice in PBS, permeabilized in PBS with 0.4% Triton X-100 for 15 min, rinsed twice in PBS and blocked in PBS with 1% BSA, 0.4% Triton X-100 for 10 min. The coverslips were incubated in PBS with anti-PAR antibody (H-10) (1:200), 1% </p><p>BSA, 0.4% Triton X-100 and 10 M ADP-HPD at 37°C for 1 h, rinsed twice in PBS, and incubated with Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (1:200) at 37°C for 30 min. Cells were rinsed twice in PBS and mounted after adding mounting medium with 4, 6 diamidino-2-phenylindole.</p><p>Preparation of cell extracts </p><p>Cells were harvested and lysed on ice for 10 min in Lysis buffer (25 mM Hepes, pH 7.5, </p><p>0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5% Triton X-100, 0.5 mM DTT and 1mM phenylmethylsulfonyl fluoride). PARP-1 was released into the supernatant (15800×g, </p><p>4°C, 10 min). The pellet was resuspended in 1×SDS loading buffer (50 mM Tris-HCl, pH</p><p>6.8, 100 mM DTT, 2% SDS, 10% glycerol) and sonicated prior to SDS-PAGE analysis of histones.</p><p>Immunoblotting and immunopreciptation</p><p>Fifty µg of cell extracts were resolved by 6% SDS-PAGE and transferred onto PVDF. </p><p>Following antibody incubation, membranes were developed by chemiluminescence using a peroxidase coupled secondary antibody. For the immunoblotting of  H2AX , 10 µg of proteins from cell pellet were resolved by 15% SDS-PAGE. For immunoprecipitation, </p><p>500 µg of proteins in 500 µl of IP buffer (12.5 mM Hepes, pH 7.5, 150 mM NaCl, 0.75 mM MgCl2, 0.1 mM EDTA, 0.25% Triton X-100, 0.25 mM DTT and 1mM phenylmethylsulfonyl fluoride) were incubated with 1.5 µg of anti-Ku70/80 monoclonal antibody (Ku Ab-3) or 10 µl of anti-PARP-1 antibody at 4 °C for 2 hr. Immunoprecipitates were recovered with protein G or protein A sepharose beads, washed five times with IP buffer, and analyzed by SDS-PAGE. Input lanes correspond to 10% of immunoprecipitated material.</p><p>ADP-ribosylation </p><p>Fifty µg of cell extract in 50 µl of IP buffer with 10 µM ADP-HPD (Calbiochem, La </p><p>Jolla, CA) was incubated with 1 µCi of [32P] adenylate NAD+ (~1000 Ci/mmol) </p><p>(Amersham Biosciences, ) at 30 °C for 10 min. Reactions were terminated with SDS loading buffer, resolved by SDS-PAGE, PVDF transfer and autoradiography. For experiments with protein kinase and protein phophatase inhibitors, additional assays were employed to ensure the functionality and specificity of the inhibitors.</p><p>Protein dephosphorylation by Calf Intestinal Alkaline Phosphatase (CIP) and 2-D analysis</p><p>Cell extracts (150 µg/ 100 µl) were incubated with 10 units of Calf Intestinal Alkaline </p><p>Phosphatase or phosphatase inhibitors at 37 °C for 30 min, and acetone precipitated. </p><p>Pellets were solubilized in 2-D rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, </p><p>1% DTT) by sonication, adjusted to 0.5% Biolytes 3-10 (Bio-Rad) and subjected to isoelectric focusing on a 7-cm IPG strip of pH 3-10 NL (Amersham Biosciences, </p><p>Piscataway, NJ). Focused IPG strips were incubated in equilibration buffer (50 mM Tris-</p><p>HCl, pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% SDS) containing 1% DTT followed by incubation in equilibration buffer containing 4% iodoacetamide. Proteins were then resolved by 8% SDS-PAGE and transferred to PVDF. </p>