Association of TdT with 55- and 110-kDa proteins. (A) Schematic representation of TdT constructs. Full-length TdT (58 kDa) containing an N-terminal BRCT domain, a DNA-binding domain (DBD), and a nucleotide-binding domain (NBD); 44-kDa protein lacking the BRCT domain; 22-kDa protein lacking the DBD and NBD domains; BRCT domain only. (B and C) Proteins were resolved on SDS/10% PAGE gels and visualized by Coomassie staining. (B) GST pull-down of Molt-4 and Nalm-6 proteins binding to TdT constructs. Lane 1, molecular weight markers; lanes 2–5, GST or GST-fusion proteins incubated with 500 μg of Molt-4 cell extract; lanes 6–9, GST or GST-fusion proteins incubated with 500 μg of Nalm-6 cell extract; lane 10, purified TdT. (C) Binding of proteins from indicated tumor cell lines to the GST-BRCT. Five hundred micrograms of extracts were incubated with GST-BRCT protein in buffers containing 500 mM NaCl and 0.3% Triton X-100. Lane 1, molecular weight markers; lane 2, GST-BRCT alone; lanes 3–10, whole cell extracts incubated with fusion protein.

Sequence and schematic representation of the 55-kDa protein. (AUpper) Amino acid sequences of the hPso4 peptide fragments identified by mass spectrometry are underlined. (Lower) Schematic representation of the hPso4 protein showing the U-box location, Prp19 homology region, and WD-40 repeats. (B) Northern analysis of hPSO4 mRNA in human multiple tissue blots (Clontech).

Coimmunoprecipitation of TdT and hPso4. (A) Western blots of 50 μg of Molt-4 and Jurkat cell extracts by using affinity-purified rabbit polyclonal antibodies raised to the GST-Pso4 fusion protein. (B) Immunoprecipitation of TdT from 200μg of Molt-4 extract by using preimmune serum, anti-hPso4, anti-hCdc5, and anti-TdT antibodies. Immune complexes were probed by using anti-TdT antibody. Lane 1 contains 60 μg of Molt4 lysate. (C) Purification of recombinant hPso4 from Sf9 cells. (Upper) Coomassie stain. Lane 1, molecular weight markers; lane 2, input; lane 3, FT flow-through; lanes 4 and 5, wash; lanes 6–10, 100 mM imidazole eluates. (Lower) Western blot by using anti-His antibodies. (D) Interaction of TdT and hPso4. Lane 1, purified TdT; lane 2, purified TdT with anti-His antibody; 58-kDa TdT immunoprecipitated from a preformed complex of 44-kDa TdT, 58-kDa TdT, and His-hPso4 with anti-TdT (lane 3) or anti-His antibody (lane 4).

Binding of hPso4 to DNA radiolabeled DNA was incubated with purified hPso4 protein. The reaction products were analyzed on native polyacrylamide gels followed by autoradiography (A and B) or 0.7% agarose gel stained with ethidium bromide (C). (A) hPso4 binding to ssDNA. Lane 1, ³²P-labeled 50-mer ssDNA (5 nM) and increasing amounts of hPso4 (lanes 2–6, 90–540 nM). (B) hPso4 binding to dsDNA; 5 nM ³²P-labeled 50 bp of duplex DNA (lane 1) and increasing amounts of hPso4 (lanes 2–6, 90–540 nM). (C) hPso4 binding to circular plasmid DNA; 12 nM pBKS dsDNA (lane 1) and increasing amounts of hPso4 (lanes 2–5, 0.4–0.7 μm).

Time course of hPso4 induction in response to DNA damage MRC5 cells treated with 12-Gy γ irradiation (A), 100 μg/ml bleomycin (B), 100 μg/ml cisplatin (C), 50 μg/ml mitomycin C (D), and 15 J/m² uv (E). Western blots containing 30 μg of cell extracts prepared from untreated or treated (0.5–6 h) cells were probed with anti-hPso4, antiphosphoserine15-p53 (P-serine 15), or anti-Ku86 antibodies, as indicated.

Effect of siRNA transfection on hPso4 expression. (A) MRC5 cells were transfected with control or hPso4 siRNA. Twenty-four hours posttransfection, cells were γ-irradiated at 12 Gy. Western blots containing 75 μg of cell extracts prepared from nonirradiated or irradiated (1–4 h) cells were probed with anti-hPso4 or anti-Ku86 antibodies, as indicated. (B) Western analysis of whole cell extracts prepared from HeLa cells (1.5 × 10⁶) transfected with either the control siRNA (lanes 2 and 5) or the hPso4-siRNA (lanes 3 and 6) and probed with anti-hPso4 or anti-Ku 86 antibodies. (C) Flow cytometry of control and hPso4 siRNA transfected cells unirradiated (Upper) or γ-irradiated at 15 Gy (Lower), incubated in fresh growth medium to allow repair, and fixed after 3 h. Cells were stained with anti-BrdUrd-FITC to detect DSBs at which Br-dUTP had been incorporated by using TdT (14, 19). Similar results were obtained in a second experiment.

Effect of hPso4 on cell survival after DNA damage. Cell death ELISAs were performed on 10⁵ HeLa cells or cells transfected with the control or hPso4 siRNAs and treated with etoposide (A) or mitomycin C (B) for 1 h. Each graph is the result of three independent transfections assayed in duplicate and represents the mean ± SEM. Clonogenic survival assays were performed on 10⁵ HeLa parental cells or cells transfected with the control or hPso4 siRNAs and exposed to indicated doses of γ irradiation. Cells were trypsinized and then plated at a density of 200 cells per 100-mm-diameter dish. Surviving colonies were counted after 12 days.(C) A representative clonogenic survival assay. (D) Survival data from three independent transfections assayed in triplicate. Data are shown as percent of control and represent the mean ± SEM.