The level of 8-oxoG, the most common oxidation products was significantly increased after H2O2 treatment (Fig. and accumulated more DNA damage lesions in their genome. Taken together, these results demonstrate that H4R3me2s can be recognized as a reader protein that senses DNA damage and a writer protein that promotes DNA repair. BL21DE and were purified using Ni2+ affinity chromatography (GE Healthcare). DNA Ligase I (Lig I) was purchased from Abnova. 2.4. FEN1 nuclease activity assay The nuclease activity reaction was performed in buffer made up of 50?mM Tris-HCl (pH 8.0), 50?mM NaCl and 5?mM MgCl2. The FAM-labeled DNA substrates, purified FEN1 protein and H4R3me2s or H4R3 peptide were mixed and incubated at 37?C for 30?min in the dark, stopped by addition of the stop buffer (95% formamide, 20?mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol). Finally, the combination was separated by 15% PAGE made up of 8?M urea and analyzed by Odessey FC. 2.5. Electrophoretic mobility shift assay The same concentration of FEN1 protein and various concentrations of H4R3me2s or H4R3 peptide were incubated with FAM-labeled DNA substrates in a buffer made up of 25?mM Tris/HCl (pH 8.0), 1?mM DTT, 5% glycerol, 0.25?mg/mL BSA, 50?mM NaCl and 0.2?mM EDTA. The combination was put together on ice and preincubated for 10?min. After further incubation of 10?min?at 37?C, the combination was separated by a 6% non-denaturing polyacrylamide gel (30:1 acrylamide: bis-acrylamide) in TBE buffer and imaged by Odessey FC. 2.6. Reconstituted LP-BER assay The reconstituted BER assay was performed in 20?l of reaction buffer containing 40?mM HEPESCKOH (pH 7.8), 70?mM KCI, 7?mM MgCl2, 1?mM dithiothreitol, 0.5?mM EDTA, 2?mM ATP, 200 U creatine-phosphokinase, 0.5?mM NAD and 5?mM phosphocreatine, 50?M each of dATP, dTTP and dGTP and 8?Ci [-32P]-dCTP. For the BER reaction with purified protein, FEN1 (15?ng), APE1 (2?ng), PCNA (2?ng), Ligase I (20?ng), FAM-labeled DNA substrates and various amounts of H4R3me2s or H4R3 peptide (0C2?g) was mixed on ice and incubated for 30?min?at 37?C. For the BER reaction with the whole cell extract, the dATP, dTTP, dGTP, [-32P]-dCTP and DNA substrates were incubated with cell extract at 37?C for 30 min. Reactions were stopped with stop buffer, heated at 95?C for 10?min, and 10?l of the reaction combination were separated by 15% PAGE containing 8?M urea and imaged by Odyssey FC. 2.7. Drug sensitivity assay Drug PF-06305591 sensitivity assay was measured by trypan blue exclusion analysis. Cells were seeded onto a 12-well plate in 1?ml cell culture medium of each well. After transfected with the indicated siRNA or plasmids, the cells were treated with numerous concentrations of H2O2 for 30?min and continuously cultured in fresh medium for 48?h, the cells were collected and resuspended in cell culture medium. An equal volume of 0.4% trypan blue dye was added and mixed with the cell suspension. The viable cells were measured by PF-06305591 Count Star. 2.8. Immunofluorescence Cells were cultured in 6-well plate which contained an acid-treated slide and incubated overnight. Then the slides were washed with PBS three times, treated with 4% formaldehyde in PBS for 30?min and then washed again with PBS. Triton X-100 Rabbit Polyclonal to LMO4 (0.05%) was added for 5?min to permeabilize the cells. The slides were blocked by 5% BSA and then incubated with main antibody at 4?C overnight. The slides were washed with PBS three times and then incubated with fluorescent second of all antibody conjugated with fluorescein isothiocyanate for 2?h. Then the slides were washed again with PBS and stained with DAPI for 10?min. The mounted slides were viewed with a Nikon 80I 10-1500X microscope, and images were captured with a charge-coupled-device video camera. 2.9. Streptavidin pull down assay The biotin-labeled histone peptides were synthesized by Chinese Peptide Organization, HangZhou. Cells were lysed in IP buffer made up of PMSF and cocktail inhibitor (Roche) at 4?C for 3?h. After PF-06305591 centrifugation at 12,000?rpm for 10?min?at 4?C, the supernatants were further treated with 100?g/ml DNase I for 15?min. pulldown assay was performed by incubating the histone peptides with cell lysates overnight PF-06305591 using Streptavidin beads, the next day washed the beads three times with PBS for 5?min each. The proteins that remained bound to the peptides were separated by SDS-PAGE and visualized by silver staining. 3.?Results 3.1. H4R3me2s is usually upregulated under DNA damage stress The symmetrical dimethylation of arginine-3 of histone H4 (generating H4R3me2s) is related to the repressed transcription of several genes under different stresses [20]. Oxidative stress is the most common cellular stressor and can lead to oxidatively damaged DNA. To test whether H4R3me2s is usually a response to this stress, we first chose hydrogen peroxide (H2O2), a well-known oxidizer, to treat.