FIGURE 6.

Demethylase activity of hABH1(Δ3). A, demethylase activity of hABH1(Δ3) (10 pmol), hABH3 (10 pmol), and AlkB (5 pmol) on different [³H]methylnitrosourea-methylated polymers of DNA and RNA. B, [³H]methyl-poly(dC) was incubated with AlkB or hABH1(Δ3) or with both proteins for 1 h, followed by ethanol precipitation. The ethanol-soluble radioactivity was quantified by scintillation counting. C, inhibition by polyclonal anti-hABH1 antibody of hABH1(Δ3) (15 pmol) demethylase activity using [³H]methyl-poly(dC) as substrate. AlkB (15 pmol) was used as control. D, demethylase activity of hABH1(Δ3) (20 pmol), on single-stranded oligonucleotide substrate (1 pmol) containing 3-meC, using the fluorescent oligonucleotide substrate assay. The standard reaction mixture was modified as depicted. E, HPLC elution profile of the products generated by acid hydrolysis of the ethanol-precipitated [³H]methyl-poly(dC) after incubation with AlkB or hABH1(Δ3) (10 pmol), or without enzyme. The arrow indicates retention time for unlabeled 3-meC standard as detected by UV absorbance at 260 nm. F, as in E but including whole mitochondria cell extract (MPE) (50 μg), hABH1(Δ3) (10 pmol), or without enzyme.