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. 2008 Oct 15;582(23-24):3313-9.
doi: 10.1016/j.febslet.2008.08.019. Epub 2008 Sep 5.

Oxidative demethylation of 3-methylthymine and 3-methyluracil in single-stranded DNA and RNA by mouse and human FTO

Guifang Jia  1 , Cai-Guang Yang, Shangdong Yang, Xing Jian, Chengqi Yi, Zhiqiang Zhou, Chuan He
Affiliations

Oxidative demethylation of 3-methylthymine and 3-methyluracil in single-stranded DNA and RNA by mouse and human FTO

Guifang Jia et al. FEBS Lett. .

Abstract

The human obesity susceptibility gene, FTO, encodes a protein that is homologous to the DNA repair AlkB protein. The AlkB family proteins utilize iron(II), alpha-ketoglutarate (alpha-KG) and dioxygen to perform oxidative repair of alkylated nucleobases in DNA and RNA. We demonstrate here the oxidative demethylation of 3-methylthymine (3-meT) in single-stranded DNA (ssDNA) and 3-methyluracil (3-meU) in single-stranded RNA (ssRNA) by recombinant human FTO protein in vitro. Both human and mouse FTO proteins preferentially repair 3-meT in ssDNA over other base lesions tested. They showed negligible activities against 3-meT in double-stranded DNA (dsDNA). In addition, these two proteins can catalyze the demethylation of 3-meU in ssRNA with a slightly higher efficiency over that of 3-meT in ssDNA, suggesting that methylated RNAs are the preferred substrates for FTO.

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Figures

Fig. 1
Fig. 1
A restriction enzyme digestion assay for repair of 1-meA, εA, and 1-meG by ABH2, mFTO, and hFTO. (A) A 49 mer ssDNA (0.2 nmol) with 1-meA, εA, or 1-meG incorporated into a GATC sequence (can be recognized and cleaved by DpnII) was used for the assay. The modified dsDNA probe is resistant to DpnII cleavage. Repair of the base lesion led to cleavage of the 49 mer dsDNA into two fragments of 27 and 22 base pairs, which was analyzed by a denaturing DNA gel. Both mFTO (0.2 nmol) and hFTO (0.2 nmol) exhibited very low activities toward 1-meA in ssDNA (12 h at 16°C and pH 6.0). Repair of εA and 1-meG by mFTO and hFTO were not observed. (B) No activities toward 1-meA, εA, and 1-meG in dsDNA were observed for either mFTO or hFTO under the same assay conditions. ABH2 showed good activities to repair 1-meA and εA in dsDNA in control experiments.
Fig. 2
Fig. 2
HPLC chromatograms of digested nucleosides from 3-meT-containing DNA (1 nmol), 3-meC-containing DNA (1 nmol) and 3-meU-containing RNA (1 nmol). (A) Complete demethylation of 3-meT in a 15 mer ssDNA by mFTO and hFTO. All reactions were run for 12 h at 16°C and pH 6.0. (B) Both mFTO and hFTO gave ~15% demethylation of 3-meC in 15 mer ssDNA; 100% repair of 3-meT was observed for both enzymes under the same conditions as shown in (A). (C) Negligible repair of 3-meT in dsDNA was observed for both mFTO and hFTO under the same conditions. (D) Complete demethylation of 3-meU in a 15 mer ssRNA by mFTO and hFTO under the same conditions.
Fig. 3
Fig. 3
The pH-activity profiles for demethylation reactions of 3-meT in ssDNA and 3-meU in ssRNA by mFTO and hFTO. All reactions were run in triplicate at 16°C for 12 h. (A) Demethylation of 3-meT in the 15 mer ssDNA (1 nmol) by mFTO (0.05 nmol). (B) Demethylation of 3-meU in the 15 mer ssRNA (1 nmol) by mFTO (0.1 nmol). (C) Demethylation of 3-meT in the 15 mer ssDNA (1 nmol) by hFTO (0.5 nmol). (D) Demethylation of 3-meU in the 15 mer ssRNA (1 nmol) by hFTO (0.2 nmol).
Fig. 4
Fig. 4
Kinetics of demethylation reactions catalyzed by mFTO and hFTO. All reactions were run in triplicate at 20°C and pH 6.0. (A) Demethylation of 3-meT in the 15 mer ssDNA by mFTO (0.5 μM). (B) Demethylation of 3-meU in the 15 mer ssRNA by mFTO (0.3 μM). (C) Demethylation of 3-meT in the 15 mer ssDNA by hFTO (2.5 μM). (D) Demethylation of 3-meU in the 15 mer ssRNA by hFTO (0.4 μM).
Fig. 5
Fig. 5
FTO can demethylate both 3-meT from ssDNA and 3-meU from ssRNA.
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