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AlkB reverses etheno DNA lesions caused by lipid oxidation in vitro and in vivo

Abstract

Oxidative stress converts lipids into DNA-damaging agents. The genomic lesions formed include 1,N6-ethenoadenine (εA) and 3,N4-ethenocytosine (εC), in which two carbons of the lipid alkyl chain form an exocyclic adduct with a DNA base. Here we show that the newly characterized enzyme AlkB repairs εA and εC. The potent toxicity and mutagenicity of εA in Escherichia coli lacking AlkB was reversed in AlkB+ cells; AlkB also mitigated the effects of εC. In vitro, AlkB cleaved the lipid-derived alkyl chain from DNA, causing εA and εC to revert to adenine and cytosine, respectively. Biochemically, εA is epoxidized at the etheno bond. The epoxide is putatively hydrolyzed to a glycol, and the glycol moiety is released as glyoxal. These reactions show a previously unrecognized chemical versatility of AlkB. In mammals, the corresponding AlkB homologs may defend against aging, cancer and oxidative stress.

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Figure 1: Structures of alkylated bases and site-specific in vivo approach used.
Figure 2: Lethality and mutagenicity of εA, εC, m1G and THF.
Figure 3: AlkB-mediated direct reversal of εA and m1A in vitro.
Figure 4: AlkB-mediated direct reversal of εA in vitro.
Figure 5
Figure 6: Sensitivity of E. coli deficient in AlkA or AlkB to CAA.

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Acknowledgements

We thank T.J. Begley for initially constructing the AlkB expression vector and A.M. Herrera and A. Fichera for NMR assistance. We further acknowledge the pioneering work of the late E. Seeberg and B. Singer. We thank Agilent Technologies for access to the 1100 MSD TOF mass spectrometer and J. Marr and J. Lau of Agilent for helpful discussions. This work was supported by the US National Institutes of Health (grants CA80024, CA75576; CA55043; ES11399; P01-CA26731; GM069857 and P30-ES02109).

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Correspondence to John M Essigmann.

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Supplementary information

Supplementary Fig. 1

Schematic for lesion bypass and mutagenesis studies in vivo. (PDF 30 kb)

Supplementary Fig. 2

Schematic for quantifying fully ligated genomes for the CRAB lesion bypass assay. (PDF 22 kb)

Supplementary Fig. 3

MALDI-TOF of εA vs. εC repair by AlkB (PDF 46 kb)

Supplementary Fig. 4

ESI-TOF of εA repair by AlkB (PDF 30 kb)

Supplementary Methods (PDF 38 kb)

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Delaney, J., Smeester, L., Wong, C. et al. AlkB reverses etheno DNA lesions caused by lipid oxidation in vitro and in vivo. Nat Struct Mol Biol 12, 855–860 (2005). https://doi.org/10.1038/nsmb996

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