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. 2005 Oct 26;127(42):14594-5.
doi: 10.1021/ja055957m.

Direct repair of the exocyclic DNA adduct 1,N6-ethenoadenine by the DNA repair AlkB proteins

Yukiko Mishina  1 , Cai-Guang Yang, Chuan He
Affiliations

Direct repair of the exocyclic DNA adduct 1,N6-ethenoadenine by the DNA repair AlkB proteins

Yukiko Mishina et al. J Am Chem Soc. .

Abstract

The exocyclic DNA base adduct 1,N6-ethenoadenine (epsilonA) is directly repaired by the AlkB proteins in vitro.

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Figures

Figure 1
Figure 1
Proposed repair of εA in DNA by the AlkB proteins.
Figure 2
Figure 2
Repair of T(εA)T by E. coli AlkB. (A) A reaction of T(εA)T (0.1 mM) with E. coli AlkB (0.01 mM) under the standard repair conditions (50 mM MES or HEPES, 0.2 mM Fe(NH4)2(SO4)2, 2.6 mM α-ketoglutarate, 5.2 mM ascorbate) at pH 7.0 for 1 h at 37 °C led to the formation of TAT, which was resolved by HPLC and detected at A254. (B) A control experiment under the same conditions as (A) but in the absence of AlkB gave only the starting material T(εA)T. (C) The reaction mixture of (A) was analyzed by MALDI-TOF mass spectrometry. The negative molecular peak of TAT at 858.2 was detected. (D) The mixture of the control reaction in (B) was analyzed by MALDI-TOF mass spectrometry, and the negative molecular peak of T(εA)T at 882.2 was observed.
Figure 3
Figure 3
The pH dependency and time courses for the repair of T(εA)T by AlkB. (A) T(εA)T (0.1 mM) was treated with 5 µM of AlkB at different pH, and the reactions were quenched after 5 min. Time courses for reactions between 0.1 mM of T(εA)T and AlkB at 37 °C; (B) 5 µM AlkB at pH 6.0, (C) 5 µM AlkB at pH 7.0, and (D) 10 µM of AlkB with pH 8.0 are also shown. Standard repair conditions were used with MES (for pH 4.0–6.5) or HEPES (for pH 7.0–8.5) as the buffers in 100 µL of solution.
Scheme 1
Scheme 1
Exocyclic DNA Adducts
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