2. Academic Validation
  3. Differential repair of etheno-DNA adducts by bacterial and human AlkB proteins

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

  • DNA Repair (Amst). 2015 Jun;30:1-10. doi: 10.1016/j.dnarep.2015.02.021.
Daria Zdżalik 1,  Anna Domańska 2,  Paulina Prorok 3,  Konrad Kosicki 1,  Erwin van den Born 4,  Pål Ø Falnes 4,  Carmelo J Rizzo 5,  F Peter Guengerich 6,  Barbara Tudek 7
Affiliations

Affiliations

  • 1 Institute of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland.
  • 2 Institute of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland; Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
  • 3 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
  • 4 Department of Biosciences, University of Oslo, Oslo, Norway.
  • 5 Department of Chemistry, Center in Molecular Toxicology, and Vanderbilt Ingram Cancer Center, Vanderbilt University, Nashville, TN, USA.
  • 6 Department of Biochemistry, Center in Molecular Toxicology, and Vanderbilt Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN, USA.
  • 7 Institute of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland; Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland. Electronic address: [email protected].
PMID: 25797601 DOI: 10.1016/j.dnarep.2015.02.021
Abstract

AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno(ɛ)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of ɛ-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 ɛ-adducts, 1,N(6)-ethenoadenine (ɛA), 3,N(4)-ethenocytosine (ɛC) and 1,N(2)-ethenoguanine (1,N(2)-ɛG) 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, engaged in 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 ɛ-adducts, since they do not repair methylated bases. Three other proteins, EcAlkB (Escherichia coli), SA-1A, and XC-1B removed ɛA and ɛC from ds and ssDNA but were inactive toward 1,N(2)-ɛG. SC-1A repaired only ɛA with the preference for dsDNA. The human Enzyme ALKBH2 repaired all three ɛ-adducts in dsDNA, while only ɛA and ɛC in ssDNA and repair was less efficient in ssDNA. ALKBH3 repaired only ɛC 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,N(2)-ɛG and that ALKBH3 removes only ɛC from ssDNA. Our results also suggest that the nucleotide recognition lid is not the sole determinant of the substrate specificity of AlkB proteins.

Keywords

1,N(2)-Ethenoguanine; 1,N(6)-Ethenoadenine; 3,N(4)-Ethenocytosine; AlkB; DNA repair; Etheno adducts.

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