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Differential repair of etheno-DNA adducts by bacterial and human AlkB proteins

Zdżalik, Daria and Domańska, Anna and Prorok, Paulina and Kosicki, Konrad and van den Born, Erwin and Falnes, Päl Ø and Rizzo, Carmelo J and Guengerich, Peter F and Tudek, Barbara (2015) Differential repair of etheno-DNA adducts by bacterial and human AlkB proteins. DNA Repair, 30 . pp. 1-10. ISSN 1568-7864

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Official URL: http://www.sciencedirect.com/science/article/pii/S...


AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno(ε)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of ε-adducts by AlkB enzymes using oligodeoxynucleotides with a single lesion and specific DNA glycosylases and AP-endonuclease for identification of the repair products. We compared the repair of three ε-adducts, 1,N6-ethenoadenine (εA), 3,N4-ethenocytosine (εC) and 1,N2-ethenoguanine (1,N2-εG) by nine bacterial and two human AlkBs, representing four different structural groups defined on the basis of conserved amino acids in the nucleotide recognition lid, responsible for the enzyme binding to the substrate. Two bacterial AlkB proteins, MT-2B (from Mycobacterium tuberculosis) and SC-2B (Streptomyces coelicolor) did not repair these lesions in either double-stranded (ds) or single-stranded (ss) DNA. Three proteins, RE-2A (Rhizobium etli), SA-2B (Streptomyces avermitilis), and XC-2B (Xanthomonas campestris) efficiently removed all three lesions from the DNA substrates. Interestingly, XC-2B and RE-2A are the first AlkB proteins shown to be specialized for ε-adducts, since they do not repair methylated bases. Three other proteins, EcAlkB (Escherichia coli), SA-1A, and XC-1B removed εA and εC from ds and ssDNA but were inactive towards 1,N2-εG. SC-1A repaired only εA with the preference for dsDNA. The human enzyme ALKBH2 repaired all three ε-adducts in dsDNA, while only εA and εC in ssDNA and repair was less efficient in ssDNA. ALKBH3 repaired only εC in ssDNA. Altogether, we have shown for the first time that some AlkB proteins, namely ALKBH2, RE-2A, SA-2B and XC-2B can repair 1,N2-εG and that ALKBH3 removes only εC from ssDNA. Our results also suggest that the nucleotide recognition lid is not the sole determinant of the substrate specificity of AlkB proteins.

Item Type:Article
Uncontrolled Keywords:AlkB, etheno adducts, 1,N6-ethenoadenine, 3,N4-ethenocytosine, 1,N2-ethenoguanine, DNA repair
Subjects:Q Science > Q Science (General)
Divisions:Department of Molecular Biology
ID Code:1014
Deposited By: Prof. Barbara Tudek
Deposited On:10 Nov 2015 15:28
Last Modified:08 Mar 2018 15:33

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