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  • In laboratory strains of Escherichia coli three mechanisms


    In laboratory strains of Escherichia coli, three mechanisms of repair for alkylated bases are known. The first involves constitutively expressed Ogt and inducible Ada methyltransferases, directly removing methyl groups from O6-methylguanine (O6meG) and O4-methylthymine (O4meT) [1], [2], [3], [4]. Induced AlkA (DNA glycosylase II) and Tag (DNA glycosylase I) expressed constitutively provide a second mechanism to remove N3-methyladenine (3meA) and create abasic (AP) sites in DNA, which are subsequently repaired by urokinase excision (BER) [5], [6]. The third mechanism employs AlkB dioxygenase to directly restore the natural DNA and RNA bases [7], [8]. N1-methyladenine (1meA) and N3-methylcytosine (3meC) are primary substrates for AlkB; however, structurally similar lesions, e.g., N1-methylguanine (1meG) and N3-methylthymine (3meT) in addition to bulky adducts, are also repaired [9], [10], [11]. DNA damage caused by alkylating agents induces the Adaptive (Ada) response [12], [13], which is extensively exploited by microbes. In laboratory conditions, this phenomenon is defined as the ability of microorganisms to acquire resistance in order to adapt to higher concentrations of mutagens when incubated in the presence of lower amounts of this deleterious compound. In E. coli, Ada response involves induction of four genes ada, alkA, alkB and aidB, which remain under the control of Ada protein functioning as the transcription factor. The organization and importance of the Ada response to preserve genome integrity differs between species. This diversity is exemplified in the Ada response and Ada regulon composition. In Pseudomonas putida, the AlkB protein is not induced as part of the Ada response but expressed constitutively [14], in contrast to E. coli. There are several valuable reviews on the Ada response that describe the state of knowledge in early 2000 but do not explore the variation of the Ada response among species or describe activation at the transcriptional level [13], [15], [16], [17]. In this review, we have summarized these two issues.
    DNA alkylation Alkylating agents damage DNA by introducing a methyl/ethyl group on available nitrogen and oxygen atoms in its bases. These chemicals can be divided into two subgroups, SN1 and SN2, according to their reaction mechanisms. The SN1 type, e.g., N-methyl-N-nitrosourea (MNU) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), act by a monomolecular mechanism and create both, N- and O-methylation, whereas in SN2 type, e.g., methyl methanesulfonate (MMS) and methyl iodide (MeI), react by bimolecular mechanisms resulting mainly in N-methylation (Fig. 1) [18]. Many modifications of nucleotide bases have been identified [19], [20]. Among them N7-methylguanine (7meG), 3meA and O6meG are major products of alkylation; whereas 1meA, 3meC, O4meT and methyl phosphotriesters (MPT) condense in smaller amounts. Some methylated bases, e.g., 3meA, 1meA, 3meC and 3meG, are cytotoxic because they block replication, whereas others, such as O6meG and O4meT, have mutagenic effects (Fig. 2, Fig. 3) [20]. Halocarbons are the most environmentally abundant alkylating agents. Methyl chloride (MeCl) is generated by the abiotic conversion of chloride or as a product of chloride detoxification [21]. Methyl bromide has been found in the oceans and is also generated by man, contributing to stratospheric ozone depletion [22]. The N-nitroso compounds present in tobacco smoke are recognized as environmental alkylating agents impacting human health. The most carcinogenic are considered to be 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosoamino)-1-(3-pirydyl)-1-butanol (NNAL), and N′-nitrosonornicotine (NNN) [23]. Myosamine, a tobacco alkaloid also detected in maize, rice, wheat, potato, milk and fruits may be implicated in esophageal cancer [24]. Of the endogenous alkylating agents, S-adenosylmethionine (SAM) acts through an SN2 mechanism and seems to be one of the main methyl group donors affecting DNA [25]. It mainly causes 7meG, 3meA, and O6meG lesions; however, its reactivity is about one 2000th that of MMS (Fig. 1, Fig. 2) [26]. Alterations in SAM concentration influence cellular function and may be implicated in some diseases and spontaneous carcinogenesis [27]. Other internal sources of alkyl groups originate from nitrosation of glycine and its derivatives [28], [29].