Alleviation of 1, N ⁶ -ethanoadenine genotoxicity by the Escherichia coli adaptive response protein AlkB

Abstract

1,N 6 -ethanoadenine (EA) forms through the reaction of adenine in DNA with the antitumor agent 1,3-bis(2-chloroethyl)-1-nitrosourea, a chemotherapeutic used to combat various brain, head, and neck tumors. Previous studies of the toxic and mutagenic properties of the DNA adduct EA have been limited to in vitro experiments using mammalian polymerases and have revealed the lesion to be both miscoding and genotoxic. This work explores lesion bypass and mutagenicity of EA replicated in vivo and demonstrates that EA is neither toxic nor mutagenic in wild-type Escherichia coli. Although the base excision repair glycosylase enzymes of both humans and E. coli possess a weak ability to act on the lesion in vitro, an in vivo repair pathway has not yet been demonstrated. Here we show that an enzyme mechanistically unrelated to DNA glycosylases, the adaptive response protein AlkB, is capable of acting on EA via its canonical mechanism of oxidative dealkylation. The reaction alleviates the unrepaired adduct's potent toxicity through metabolism at the C8 position (attached to N1 of adenine), producing a nontoxic and weakly mutagenic N 6 adduct. AlkB is shown here to be a geno-protective agent that reduces the toxicity of DNA damage by converting the primary adduct to a less toxic secondary product.

Comprehensive Assessment of Oxidatively Induced Modifications of DNA in a Rat Model of Human Wilson's Disease

“…However, results from our in vitro biochemical assay suggested Aside from TET enzymes, other members of the Fe(II)-and 2-OG-dependent dioxygenase family may also act as potential molecular targets for copper toxicity. Escherichia coli AlkB protein, which is an ortholog of human ALKBH2 and ALKBH3, were shown to be capable of repairing etheno DNA lesions (46,47). Thus, compromised ALKBH repair activity arising from elevated copper content may also account, in part, for greater accumulation of etheno DNA lesions in liver tissues of LEC rats relative to LEA rats.…”

The Aminoglycoside Antibiotic Kanamycin Damages DNA Bases in Escherichia coli: Caffeine Potentiates the DNA-Damaging Effects of Kanamycin while Suppressing Cell Killing by Ciprofloxacin in Escherichia coli and Bacillus anthracis

“…For example, AlkB was initially characterized as a dioxygenase resulting in the removal of the lesions 1-methyladenine and 3-methylcytosine, and its action was subsequently found to extend to 3-alkylpyrimidines and 1-alkylpurines (21,24,42). However, Essigmann and coworkers have shown that AlkB is the principal enzyme that removes 1,N 6 -ethenoadenine and 1,N 6 -ethenocytosine (20) as well as 1,N 6 -ethanoadenine (28), adducts that can result from hydroxyl radical lipid peroxidation-mediated DNA damage (25). Other glycosylases that remove oxidized bases include AlkA (7,46), MUG (37,59), KsgA (71), and endonucleases III (55,62) and VIII, encoded by the nth gene (33) and the nei gene (36,48), respectively.…”

The AlkB Family of Fe(II)/α-Ketoglutarate-dependent Dioxygenases: Repairing Nucleic Acid Alkylation Damage and Beyond

“…After the initial oxidation, the exocyclic bridge can open, forming another monoalkyl lesion that either can fall apart spontaneously or is further oxidized by AlkB. For example, 1,N 6 -ethanoadenine (EA), the saturated analog of ⑀A, is completely repaired by E. coli AlkB, with the reaction involving two successive oxidation steps at the carbons on the N1 and N 6 positions of adenine (83,88). Similarly, 3,N 4 -␣-hydroxyethanocytosine (the hydrated version of ⑀C) and the three-carbon bridge analog 3,N 4 -␣-hydroxypropanocytosine are also good AlkB substrates, but require only one oxidation at the carbon attached to the N3 of cytosine (10,84).…”