Cell culture and transfection
Human embryonic kidney (HEK) 293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Welgene, Daegu, South Korea), supplemented with 10 % fetal bovine serum (FBS; JR Scientific, Woodland, CA, USA) and 1 % penicillin–streptomycin solution (Welgene) at 37 °C in a 5 % CO2 incubator. For transfection, cells were seeded (3 × 105–6 × 105 cells/mL) and transiently transfected with Lipofectamine® 2000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. After incubating the cells for 24 h, 20 mM HU was added, and the cells were incubated for an additional 1 h.
siRNA construction
The siRNAs used to knockdown of endogenous hMYH and hTopBP1 were designed and purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). An siRNA sequence corresponding to nucleotides 415–439 of green fluorescent protein (GFP) was used as a negative control (Santa Cruz). hTopBP1 and hMYH knockdown was performed according to the manufacturer’s instructions.
Chromatin fractionation
HEK293 cells were lysed with lysis buffer (10 mM HEPES pH 7.4, 10 mM KCl, 0.05 % NP-40), and nuclear extracts were lysed in Low salt buffer (10 mM Tris–HCl pH 7.4, 0.2 mM MgCl2). Chromatins were collected by centrifugation and chromatin binding proteins were disassociated from chromatin with 0.2 N HCl, and 1 M Tris–HCl pH 8.0 was added. Chromatin binding proteins were quantified using the Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA, USA).
GST pull-down assay
E. coli BL21 cells (Real Biotech Corporation, Banqiao, Taipei, Taiwan) harboring the expression vectors (pGEX4T1/GST-hTopBP1, pET-28α/His-hMYH) were cultured and induced using isopropylthiogalactoside (IPTG, a final concentration of 0.5 mM) and incubated for 16 h at 18 °C. Cells were harvested and lysed by sonication in lysis buffer [20 mM Tris–Cl (pH 7.5), 500 mM NaCl] together with protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). After centrifugation, 5 mg/ml of supernatant were pulled down by glutathione Sepharose 4B beads (GE Healthcare, UK) or Ni–NTA agarose resin (Qiagen, Valencia, CA, USA). Ni–NTA agarose beads bound His-hMYH protein were released from resin by elusion buffer [20 mM Tris–Cl (pH 7.4), 500 mM NaCl] together with various concentration of Imidazole (50–300 mM). Equal concentration of purified His-hMYH (1.95 μg/μl) was then incubated with GST or GST-hTopBP1 in phosphate-buffered saline (PBS; Sigma-Aldrich), and proteins were incubated for 16 h at 4 °C. After incubation, beads was washed with PBS then resuspended with reduced glutathione buffer [50 mM Tris–Cl (pH 8.0), 10 mM reduced glutathione]. Mixture of beads and reduced glutathione buffer were incubated for 15 min and centrifuged, then supernatants were collected and analysed by western blot using His and GST antibody (Santa Cruz).
Western blot analysis
HEK293 cells were harvested and lysed with a lysis buffer [50 mM Tris–HCl (Bio Basic, Markham, Canada), 10 g/mL PMSF, 100 mM NaCl, 5 mM EDTA, 1 % Nonidet P-40, and protease and phosphatase inhibitor cocktail (Sigma-Aldrich)] for 1 h at 4 °C. Protein extracts were collected after centrifugation, and proteins were quantified. Protein samples were separated on an 8 or 12 % SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (PALL Corporation, NY, USA). The membranes were blocked using 3 % non-fat dry milk (Bio Basic) in Tris-buffered saline with 0.05 % Tween 20 (TBST) for 30 min. The membranes were then incubated with primary antibodies against hMYH (Abnova, Taipei, Taiwan), hTopBP1 (Abcam, Cambridge, UK), hRad9 (Bethyl Laboratories, Montgomery, TX, USA), c-Myc, GST, phospho-Chk1, Chk1, Cdc25A, phospho-Cdk2, Cdk2, and β-actin (Santa Cruz). After incubation with the primary antibodies, the membranes were incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz). Protein bands were detected with the ECL® Enhanced Chemiluminescence (ECL) analysis system (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. The intensity of western blot bands was quantified with Lab Works software (UVP Inc., Upland, CA, USA). Experiments were performed for three times and statistical analysis was conducted using student’s t test in Microsoft Excel for measuring the significance between the different values. Data were expressed as means ± standard errors and p < 0.05 (paired two-tailed t test, p < 0.01, p < 0.001) was considered statistically significant.
Immunoprecipitation
Total cell lysates were prepared from cells that were transiently transfected with different sets of expression vectors as described previously. Immunoprecipitation (IP) was conducted by incubating the lysates with anti-c-Myc, anti-GST antibodies (as indicated) for 1 h, and then adding protein A/G PLUS-agarose beads (Santa Cruz). Protein-bead complexes were pelleted by centrifugation and washed with PBS. The immunoprecipitated samples were analyzed by SDS-PAGE, and the immunoblot (IB) analysis was conducted with the indicated antibodies. To assess the endogenous hTopBP1-hMYH and hTopBP1-hRad9 interactions, IPs were conducted using the ImmunoCruz™ IP/WB Optima system (Santa Cruz). Beads were mixed with 1 μg of anti-hTopBP1 or anti-hRad9 antibodies in PBS. Then, the mixture was incubated on a rotator at 4 °C for 3 h. After incubation, the bead-antibody mixture was washed in PBS three times. Cell lysates were quantified, mixed with the bead-antibody mixture, and rotated for 16 h at 4 °C. The mixture was washed with PBS three times and then mixed with 1× loading dye. Samples were boiled, and the supernatant was collected via centrifugation.
Immunofluorescence staining
HEK293 cells were seeded (1 × 105 cells/mL) on coverglass bottom dishes (SPL Life Sciences, Pocheon, South Korea), grown for 24 h, and then treated with various concentrations of HU for 1 h. Cells were fixed with 4 % paraformaldehyde in PBS for 20 min and permeabilized with 0.1 % Triton X-100 in PBS for 10 min. Blocking was performed with 15 % FBS in PBS for 15 min at 37 °C. Cells were incubated with the indicated primary antibodies at 37 °C for 30 min, washed three times with PBS, and then incubated with Alexa Fluor® 488-conjugated anti-mouse IgG (Invitrogen) and Cy3-conjugated anti-rabbit IgG (Sigma-Aldrich) for 30 min at 37 °C. Cells were washed three times with PBS, and the nucleus was counterstained with To-Pro®-3 (Invitrogen). Finally, the cells were analyzed using a confocal fluorescence microscope (Olympus FV-1000; software, Olympus FluoView; Olympus, Center Valley, PA, USA).
Plasmid construction
The hTopBP1 expression vector was constructed as follows: cDNA fragments harboring the D1 (amino acids 1–444; BRCT 1–3), D2 (amino acids 444–991; BRCT 4–6), D3 (amino acids 991–1259; AD), and D4 (amino acids 991–1486; AD and BRCT 7–8) regions of hTopBP1 were generated by PCR. The PCR products were digested with BamH1 and Not1 and subsequently ligated into a pEBG vector. The full-length hMYH construct was generated using PCR. The PCR products were cleaved with EcoR1 and Sal1 and ligated into a pCMV-tag3A vector.