The Escherichia coli AlkB protein protects human cells against alkylation-induced toxicity

Abstract

Escherichia coli can ameliorate the toxic effects of alkylating agents either by preventing DNA alkylation or by repairing DNA alkylation damage. The alkylation-sensitive phenotype ofE. coli alkB mutants marks the alkB pathway as an extremely effective defense mechanism against the cytotoxic effects of the SN2, but not the SN1, alkylating agents. Although it is clear that AlkB helps cells to better handle alkylated DNA, no DNA alkylation repair function could be assigned to the purified AlkB protein, suggesting that AlkB either acts as part of a complex or acts to regulate the expression of other genes whose products are directly responsible for alkylation resistance. However, here we present evidence that the provision of alkylation resistance is an intrinsic function of the AlkB protein per se. We expressed the E. coli AlkB protein in two human cell lines and found that it confers the same characteristic alkylation-resistant phenotype in this foreign environment as it does in E. coli. AlkB expression rendered human cells extremely resistant to cell killing by the SN2 but not the SN1 alkylating agents but did not affect the ability of dimethyl sulfate (an SN2 agent) to alkylate the genome. We infer that SN2 agents produce a class of DNA damage that is not efficiently produced by SN1 agents and that AlkB somehow prevents this damage from killing the cell.The alkylation of DNA nitrogens and oxygens causes cell death and mutation in Eschenchia coli and in every cell type studied to date (8,19). The commonly studied alkylating agents, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methylnitrosourea (MNU), methyl methanesulfonate (MMS), and dimethyl sulfate (DMS), all produce the same collection of a dozen different alkylated lesions in DNA (32). However, because these agents alkylate DNA via different mechanisms, they produce the lesions in different proportions (18). MMS and DMS react via a SN2 mechanism and alkylate predominantly at DNA base nitrogens, producing very little alkylation at the oxygens in DNA bases and the oxygens in the sugarphosphate backbone. In contrast, MNNG and MNU react via an SN1 mechanism and efficiently alkylate both nitrogens and oxygens in DNA (7, 32).E. coli has several pathways that specifically ameliorate the toxic and mutagenic effects of DNA alkylation damage, and the absence of these pathways renders cells specifically sensitive to alkylating agents. Two DNA methyltransferases (MTases) encoded by the ada and ogt genes transfer methyl groups from various oxygens in DNA to an active-site cysteine residue in each of the MTase proteins. The Ada and Ogt MTases transfer methyl groups from 06-methylguanine (O6MeG) and 04-methylthymine (O4MeT); since O6MeG and O4MeT mispair during DNA replication, their repair protects E. coli from alkylation-induced mutation (19,25). The Ada MTase also transfers methyl groups from methylphosphotriester DNA lesions to a second active-site cysteine residue, whereupon the Ada protein is transformed into an efficient transcriptional activator for the ada, alkA4...

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

“…The research during this time included the finding of the resistance of AlkB to the treatment of the S N 2 type alkylating agents [15,16], which suggested its involvement in protecting bacteria from the lethal effects of alkylation damage [15][16][17][18]. Results from a sequence alignment study of protein fold and sequence homology on AlkB [19] placed AlkB into the α-ketoglutarate (αKG)-and Fe II -dependent dioxygenase family, a group of proteins that utilize Fe II to activate dioxygen and perform oxidation of various substrates.…”

“…These non-coding lesions probably arise in vivo at DNA replication forks and transcription bubbles where they block the progression of DNA and RNA polymerases [32]. Thus, repair of these lesions is critical, as failure to repair these lesions in DNA leads to cell death in both E. coli and human cells [16]. …”

Recent progress on the Ada response for inducible repair of DNA alkylation damage

“…Although the function of the AlkA protein was soon elucidated, the role of AlkB was only recently resolved (Dinglay et al, 2000;Falnes et al, 2002;Trewick et al, 2002). Evidence that AlkB acts alone came from its ability to confer MMS resistance to human cells (Chen et al, 1994). Also, the observation that AlkB repairs methylated single-stranded DNA in vivo predicted that 1-meA and 3-meC were its substrates (Dinglay et al, 2000).…”

Reversal of DNA alkylation damage by two human dioxygenases

“…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).…”