AlkB In Vitro Demethylation Assay.

AlkB demethylation activity was measured by two previously described methods (29, 30). For both methods, 40 µg of chemically synthesized oligonucleotides (IDT) that contain the restriction site for the methylation sensitive restriction enzyme MboI (underlined) was used (5′-GGATGCCTTC GACACCTAGC TTTGTTAGGT CTGGATCCTC GAAATACAAA GATTGTACTG AGAGTGCACC-3′). The oligonucleotides were treated with MMS buffer (5% [vol/vol] MMS and 50% EtoH in a final volume of 500 µl in presence of 200 mM K₂HPO₄ at room temperature for 16 h), dialyzed in Tris EDTA (TE) buffer (10 mM Tris pH 8.0 and 1 mM ethylenediaminetetraacetic acid (EDTA) pH 8.0 [Sigma-Aldrich E5134-50G]) using Spectra/Por dialysis membrane (molecular weight cut-off: 3,500), precipitated with 2 vol of ice-cold 100% EtoH and 0.3 M sodium acetate pH 5.5, washed twice with 70% EtoH, and dissolved in DNase/RNase free water.

AlkB was expressed in E. coli BL21 and immunoprecipitated using FLAG^® Immunoprecipitation Kit (Sigma-Aldrich) according to manufacturer’s instructions, and Flag-AlkB proteins were eluted using a Flag peptide.

This assay was performed as previously described (30). Repair reactions (50 µl) were carried out at 37°C for 1 h in the presence of 2.5 µM AlkB and 0.5 µg (1 µM) methylated oligonucleotide and reaction buffer (20 mM Tris–HCl pH 8.0, 200 µM indicated metabolite [αKG acid, succinic acid, fumaric acid, and malic acid], 2 mM L-Ascorbate, and 20 µM Fe(NH₄)₂(SO₄)₂). Formaldehyde release was detected by mixing demethylation repair reaction product with 40 µl of 5 M ammonium acetate and 10 µl of 0.5 M acetoacetanilide to make the final volume 100 µl. The fluorescent compound was allowed to develop at room temperature for 15 min, and then the entire reaction mixture was transferred to 96-well microplate and analyzed using a multimode reader setting the excitation wavelength at 365 nm and emission wavelength at 465 nm. Formaldehyde standard curve was prepared by selecting a range of pure formaldehyde concentrations from 2 to 20 µM.

This assay was performed as previously described (29). Repair reactions (50 µl) were carried out at 37 °C for 4 h in the presence of 2.5 µM AlkB and 0.5 µg (1 µM) methylated oligonucleotide and reaction buffer (20 mM Tris–HCl pH 8.0, 200 µM indicated metabolite, 2 mM L-Ascorbate, and 20 µM Fe(NH₄)₂(SO₄)₂). Repair reaction was annealed to equimolar complimentary ssDNA to generate methylated dsDNA in annealing buffer (50 mM Hepes pH 8 and 10 mM EDTA pH 8) at 37 °C for 60 min. Repaired dsDNA was digested by MboI restriction enzyme (2 h at 37 °C followed by heat inactivation). Digestion products were dissolved on 3% agarose containing SafeU (SYBR Safe DNA Gel Stain, Thermo Fisher Scientific, #{"type":"entrez-protein","attrs":{"text":"S33102","term_id":"420481","term_text":"pir||S33102"}}S33102) with 10 mM sodium borate as electrophoresis buffer at 300 V for 20 min and visualized using the Gel Documentation System (Bio-Rad).