OGG1 cleavage assay and mass spectrometry analysis

OGG1 substrate was a duplex of a 5′ IRDye800CW-labeled oligonucleotide synthesized by Bio-Synthesis (Lewisville, TX) with an 8-oxoG at position 17 (5′ IRDye800CW-ATA CGCATATACCGCTXTCGGCCGATCTCCGAT-3′, X = 8-oxoG), and a non-labeled complementary oligonucleotide (5′-ATCGGAGATCGGCCGACAGCGGTATATGCGTAT-3′). Hippocampal nuclear fractions were extracted using the Subcellular Protein Fractionation Kit for Tissue following the manufacturer’s protocols (Thermo Scientific, 87790) and diluted to 1.5 μg/μL with nuclear extraction buffer supplied in the kit. Hippocampal nuclear extracts (22.5 μg) were incubated with OGG1 substrate at 37 °C for 4 h with agitation in a 20-μL reaction (100 fmol OGG1 substrate, 20 mM Tris-HCl pH 8.0, 1 mM EDTA, 200 mM NaCl, 1 mg/mL BSA, 5% glycerol). After the reaction, samples were denatured in loading dye (95% formamide, 18 mM EDTA, 0.025% SDS, 0.025% bromophenol blue, 0.025% xylene cyanol) and loaded onto a 20% polyacrylamide gel containing 8 M urea at 200 V for 45 min. Gels were visualized using the LI-COR Odyssey imaging system, and OGG1 cleavage activity was determined by the amount of cleaved product using the ImageJ software (version 1.51m9). Samples after in vitro acetylation and deacetylation assays (described above) were incubated with OGG1 substrate (100 fmol) at 37 °C for 1 h. Heat-inactivated HDAC1 (80 °C for 20 min) and pharmacologically inhibited HDAC1 (incubated with 100 nM trichostatin A) were used to assess the stimulatory effect of HDAC1 deacetylase activity on OGG1 activity. For mass spectrometry analysis, recombinant OGG1 was incubated alone, with p300 or p300 followed by HDAC1. All three reactions were subsequently subjected to protein electrophoresis. Gel bands containing OGG1 were excised and were enzymatically digested. The resulting peptides were chromatographically separated and introduced into Orbitrap Elite mass spectrometer (Thermo Fisher) where peptides were mass measured within 3 p.p.m. and subsequently fragmented in a low-resolution ion trap.