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Iron-catalysed oxidation intermediates captured in a DNA repair dioxygenase

Abstract

Mononuclear iron-containing oxygenases conduct a diverse variety of oxidation functions in biology1,2, including the oxidative demethylation of methylated nucleic acids and histones3,4. Escherichia coli AlkB is the first such enzyme that was discovered to repair methylated nucleic acids5,6, which are otherwise cytotoxic and/or mutagenic. AlkB human homologues are known to play pivotal roles in various processes7,8,9,10,11. Here we present structural characterization of oxidation intermediates for these demethylases. Using a chemical cross-linking strategy12,13, complexes of AlkB–double stranded DNA (dsDNA) containing 1,N6-etheno adenine (εA), N3-methyl thymine (3-meT) and N3-methyl cytosine (3-meC) are stabilized and crystallized, respectively. Exposing these crystals, grown under anaerobic conditions containing iron(II) and α-ketoglutarate (αKG), to dioxygen initiates oxidation in crystallo. Glycol (from εA) and hemiaminal (from 3-meT) intermediates are captured; a zwitterionic intermediate (from 3-meC) is also proposed, based on crystallographic observations and computational analysis. The observation of these unprecedented intermediates provides direct support for the oxidative demethylation mechanism for these demethylases. This study also depicts a general mechanistic view of how a methyl group is oxidatively removed from different biological substrates.

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Figure 1: Oxidative repair of damaged nucleic acid bases by AlkB.
Figure 2: Intermediates trapped during in crystallo oxidation of εA and 3-meT.
Figure 3: A zwitterionic intermediate 3 is proposed for the demethylation of 3-meC.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates are deposited in the Protein Data Bank under accessionnumbers 3O1M,3O1O,3O1P, 3O1R,3O1S,3O1T,3O1Uand3O1V.

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Acknowledgements

This study was supported by the National Institutes of Health (GM071440 to C.H.; GM084028 to Q.C.), beamlines 23ID-B (General Medicine and Cancer Institutes Collaborative Access Team (GM/CA-CAT)), 19BM-D (Structual Biology Center (SBC-CAT)), 14BM-C (BioCARS) and 21ID-D (Life Sciences Collaborative Access Team (LS-CAT)) at the Advanced Photon Source at Argonne National Laboratory, the National Institutes of Health and the United States Department of Energy. Computational resources from the National Center for Supercomputing Applications at the University of Illinois and the Center of High Throughput Computing at UW–Madison are appreciated. We also thank X. Yang, Z. Ren and E. Duguid for crystallographic discussions.

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C.Y., G.J. and C.H. designed the experiments. Experiments were performed by C.Y., G.J., Q.D., W.Z., G.Z., X.J. and C.-G.Y.; computational analyses were performed by G.H. and Q.C. C.Y. and C.H. wrote the paper and G.H., Q.D. and Q.C. contributed to editing the manuscript.

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Correspondence to Chuan He.

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The authors declare no competing financial interests.

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This file contains Supplementary Tables 1-2, Supplementary Figures 1-22 with legends, Supplementary Notes and additional references. (PDF 10517 kb)

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Yi, C., Jia, G., Hou, G. et al. Iron-catalysed oxidation intermediates captured in a DNA repair dioxygenase. Nature 468, 330–333 (2010). https://doi.org/10.1038/nature09497

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