ALKBH enzymatic activity assay

DNA repair reactions were performed with the recombinant ALKBH3 proteins and oligonucleotide substrates in the reaction buffer (50 mM Hepes-KOH, 40 mM αketoglutarate, 2 mM Ascorbate, 40 mM FeSO4) for 1 hour at 37°C. The 3meC containing oligonucleotide substrate is 5′AAAGCAAGCAGATYATTCGAAAAAGCGAAA-3′, Y = 3-meC. The complementary oligonucleotide with fluorescent tag is 5′TTTCGCTTTTTCGAATGATCTGCTTGCTTT-Cy5.5–3′. The oligonucleotide substrate and the complementary oligonucleotide were mixed and heated to 90°C for 1 minute and cooled at 1°C /minute to 4°C to anneal. The reactions were then digested with DpnII restriction enzyme and the products were separated by 20% nondenaturing TBE-PAGE gel.