Reversal of DNA alkylation damage by two human dioxygenases

Abstract

The Escherichia coli AlkB protein protects against the cytotoxicity of methylating agents by repair of the DNA lesions 1-methyladenine and 3-methylcytosine, which are generated in singlestranded stretches of DNA. AlkB is an ␣-ketoglutarate-and Fe(II)-dependent dioxygenase that oxidizes the relevant methyl groups and releases them as formaldehyde. Here, we identify two human AlkB homologs, ABH2 and ABH3, by sequence and fold similarity, functional assays, and complementation of the E. coli alkB mutant phenotype. The levels of their mRNAs do not appear to correlate with cell proliferation but tissue distributions are different. Both enzymes remove 1-methyladenine and 3-methylcytosine from methylated polynucleotides in an ␣-ketoglutarate-dependent reaction, and act by direct damage reversal with the regeneration of the unsubstituted bases. AlkB, ABH2, and ABH3 can also repair 1-ethyladenine residues in DNA with the release of acetaldehyde.A lthough single-stranded regions of DNA occur in vivo within replication forks and transcription bubbles, the susceptibility of single-stranded DNA to alkylating agents has been little investigated. The major lesions generated in single-stranded DNA are 1-alkyladenine and 3-alkylcytosine; these modification sites are protected by the complementary strand in duplex DNA (1). The 3-methylcytosine (3-meC) lesions block replication and are potentially cytotoxic (2). The Escherichia coli AlkB function counteracts toxicity by alkylating agents and its expression is induced by exposure to such agents (3, 4). Expression of E. coli AlkB in mammalian cells also confers increased resistance to alkylating agents (5). We have shown that AlkB specifically repairs alkylation damage in single-stranded DNA in vivo, and binds preferentially to single-stranded DNA in vitro (6). These results indicated that AlkB repairs 1-methyladenine (1-meA) and͞or 3-meC residues in DNA, but the reaction mechanism was unknown. In an important lead, protein fold analysis combined with weak sequence homology suggested that AlkB is a member of the family of ␣-ketoglutarate (␣KG)-and Fe(II)-dependent dioxygenases (7). These enzymes are involved in a variety of metabolic reactions; however, a fungal member of the family can perform catabolic oxidative demethylation of the free base 1-methylthymine (8). Biochemical assays with purified AlkB protein recently demonstrated that AlkB is indeed an ␣KG-and Fe(II)-dependent dioxygenase that oxidatively demethylates 1-meA and 3-meC residues in single-stranded as well as doublestranded DNA. The methyl group is released from the lesion as free formaldehyde, with accompanying regeneration of the unsubstituted base residue in DNA (9, 10).Because alkylating agents are environmental carcinogens, and also are used clinically as cytotoxic anticancer drugs, it was of interest to determine whether human cells have a counterpart to the E. coli AlkB protein. Here, we identify and characterize two human AlkB homologs encoded on different chromosomes. Materials and MethodsSingle-Stranded DNA ...

ALKBH1-Mediated tRNA Demethylation Regulates Translation

“…m 1 A has recently been discovered as another mRNA modification, in addition to m 6 A, pseudouridine, and m 5 C. The presence of m 1 A at the 5′ UTR region in mRNA has been suggested to promote translation (Dominissini et al, 2016;Li et al, 2016). ALKBH3, a DNA repair enzyme that demethylates N 1 -methyldeoxyadenosine in single-stranded DNA (Aas et al, 2003;Duncan et al, 2002) has been suggested as a potential demethylase of m 1 A in mRNA , the functional relevance of which still requires further investigations. ALKBH3 is also capable of repairing tRNA alkylation damages (Aas et al, 2003), again the biological relevance of which was not clear.…”

Oxidative dealkylation DNA repair mediated by the mononuclear non-heme iron AlkB proteins

“…AlkB repairs various modifications as shown by the survival of alkylated bacteriophage in an E. coli alkB mutant [32,37,46,60,61]. Mainly, AlkB is known for the repair of 1-meA and 3-meC.…”

“…3, and studies have confirmed that 1-ethyladenine is repaired by AlkB to form adenine [37]. The C-1 C-H group rather than the C-2 C-H group of the ethyl adduct is oxidized, forming acetaldehyde as the released product along with the repaired base [37].S N 2 alkylating epoxides can cause hydroxyalkylated DNA lesions. For instance, ethylene oxide and propylene oxide can form 1-hydroxyalkyladenine and 3-hydroxyalkylcytosine.…”

A loop matters for FTO substrate selection

“…AlkB, hABH1, hABH2 and hABH3 have a similar specificity for 1-meA and 3-meC, but they exibit different selectivity for ss or double-stranded (ds) DNA/RNA substrates. hABH2 was found to be strikingly more active on dsDNA than on ssDNA (Duncan et al, 2002;Aas et al, 2003). By contrast, hABH1 and hABH3 strongly prefer ssDNA/RNA as substrates (Duncan et al, 2002;Aas et al, 2003;Westbye et al, 2008), whereas AlkB has a slightly higher activity toward ss than ds nucleic acids (Falnes et al, 2002).…”

“…Eight AlkB homologs are predicted to exist in human (Kurowski et al, 2003). Removal of the methyl group from 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) through oxidation by AlkB family protein has been experimentally demonstrated for E. coli AlkB (Falnes et al, 2002) and its human homologs hABH1 (Westbye et al, 2008;Müller et al, 2010), hABH2 (Duncan et al, 2002;Aas et al, 2003) and hABH3 (Duncan et al, 2002). In addition to DNA, RNA can also act as substrates of some AlkB members like AlkB, hABH1 and hABH3 (Aas et al, 2003;Westbye et al, 2008).…”