Exocyclic Carbons Adjacent to the N[superscript 6] of Adenine are Targets for Oxidation by the Escherichia coli Adaptive Response Protein AlkB

نویسندگان

  • Deyu Li
  • James C. Delaney
  • Charlotte M. Page
  • Xuedong Yang
  • Alvin S. Chen
  • Cintyu Wong
  • Catherine L. Drennan
  • John M. Essigmann
چکیده

The DNA and RNA repair protein AlkB removes alkyl groups from nucleic acids by a unique ironand αketoglutarate-dependent oxidation strategy. When alkylated adenines are used as AlkB targets, earlier work suggests that the initial target of oxidation can be the alkyl carbon adjacent to N1. Such may be the case with ethano-adenine (EA), a DNA adduct formed by an important anticancer drug, BCNU, whereby an initial oxidation would occur at the carbon adjacent to N1. In a previous study, several intermediates were observed suggesting a pathway involving adduct restructuring to a form that would not hinder replication, which would match biological data showing that AlkB almost completely reverses EA toxicity in vivo. The present study uses more sensitive spectroscopic methodology to reveal the complete conversion of EA to adenine; the nature of observed additional putative intermediates indicates that AlkB conducts a second oxidation event in order to release the twocarbon unit completely. The second oxidation event occurs at the exocyclic carbon adjacent to the N atom of adenine. The observation of oxidation of a carbon at N in EA prompted us to evaluate N-methyladenine (m6A), an important epigenetic signal for DNA replication and many other cellular processes, as an AlkB substrate in DNA. Here we show that m6A is indeed a substrate for AlkB and that it is converted to adenine via its 6-hydroxymethyl derivative. The observation that AlkB can demethylate m6A in vitro suggests a role for AlkB in regulation of important cellular functions in vivo. ■ INTRODUCTION Nuclear and mitochondrial DNA is damaged by radiation, by organic and inorganic chemical agents and by the misdirected activity of enzymes. Exogenous and endogenous processes that contribute to genomic damage include oxidation, alkylation, and deamination of DNA. Adducts arising from nucleic acid damage may cause mutations, jeopardize epigenetic patterns, block DNA and RNA synthesis, inhibit and alter the coding of mRNA transcription and translation, and promote strand breaks. Many chemical anticancer agents have been designed to form toxic DNA adducts. One example, BCNU (1,3-bis(2chloroethyl)-1-nitrosourea), has been used to treat lymphoma, multiple myeloma, and several types of brain cancer. Among the toxic DNA adducts formed by this chemotherapeutic agent is 1,N-ethanoadenine (EA, Figure 1a), which blocks DNA replication. In the EA DNA adduct, the exocyclic Nnitrogen of adenine is connected to the N1 ring nitrogen by a saturated two-carbon bridge, creating a five-membered ring involving the N1 and N atoms that otherwise would be involved in canonical Watson−Crick H-bonding (Figure 1a). EA is repaired to some extent by the Escherichia coli repair protein 3-methyladenine DNA glycosylase (AlkA) and the human alkyladenine DNA glycosylase MPG (also called AAG, ANPG, or APNG). However, for both of the enzymes, the excision of EA is far less efficient than that of the structurally related 1,N-ethenoadenine (eA), in which the two-carbon bridge is unsaturated (Figure 1d). Because EA lacks the structural features needed to form a Watson−Crick base pair with thymine, it is likely to be both toxic and mutagenic. In the absence of repair, EA blocks polymerase bypass and miscodes during attempted replication by mammalian DNA polymerases in vitro. The analogous eA adduct, which is also missing the identical base-pairing capabilities, is both toxic and mutagenic in E. coli in the absence of protection by the AlkB repair enzyme. Previously, we reported that AlkB can effectively alleviate the toxicity of the EA lesion in vivo; in AlkB-proficient cells, EA is easily tolerated and not significantly mutagenic. However, EA is extremely toxic to AlkB-deficient cells, showing an 86% reduction in replication. Preliminary in vitro experiments done in parallel show that AlkB could only partially convert EA to generate an open-ring intermediate 2 (Figure 1a, box), trappable with PFBHA. In an attempt to reconcile the potent ability of the protein to suppress toxicity in vivo with only partial conversion of the lesion in vitro, we speculated that intermediate 2, via flexible bond rotation, can take on a structure to form a regular Watson−Crick base pair with thymine (Figure 1a, box). That restructured base pair would not block DNA replication and hence enables tolerance

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Exocyclic Carbons Adjacent to the N6 of Adenine are Targets for Oxidation by the Escherichia coli Adaptive Response Protein AlkB

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تاریخ انتشار 2012