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  • AAG has a broad substrate specificity and besides methyladen

    2020-07-06

    AAG has a broad substrate specificity and besides 3-methyladenine can excise other altered purine residues, such as the minor lesions hypoxanthine and 1, N6-ethenoadenine from DNA. AAG initially activates these neutral base lesions by protonation of the base to allow for general 66 4 catalysis [16]. Of these three lesions, 3meA in DNA is the preferred AAG substrate and is released more rapidly than either hypoxanthine or 1, N6-ethenoadenine [17]. Studies using oligonucleotides containing a labile 3-methyldeoxyadenosine residue have not been performed due to technical problems in preparing such a substrate. Of the multiple DNA glycosylases that occur in certain bacteria, some share the broad substrate specificity of human AAG whilst others are absolutely specific for 3meA [18]. A DNA glycosylase that releases hypoxanthine or 1, N6-ethenoadenine but not 3meA from DNA has not been detected in bacteria or eukaryotic cells. AAG discriminates against normal purines due to unfavourable interactions with their exocyclic amino groups [19]. Nevertheless, AAG can catalyze slow excision of undamaged adenine and guanine from DNA and highlights the broad substrate range of AAG [20]. This reaction rate is so slow that it is of little relevance in vivo when compared with the continuous loss of adenine and guanine from DNA by nonenzymatic hydrolytic depurination at 37°C. However, overexpression of human AAG in S. cerevisiae results in a small increase in mutagenesis which may be due to the enhanced excision of normal bases and generation of mutagenic abasic sites [21]. Protein–protein interactions have been described between AAG and the transcriptional repressor MBD1 (5meC-methylated DNA-binding domain 1) [22], and between AAG and the nuclear transcription factor, estrogen receptor α [23]. Thus, the AAG repair enzyme may have an additional role as a co-regulator of certain transcription processes. Conversely, the interaction of AAG with transcription factors could affect the availability of AAG to initiate base excision-repair. AAG bound to partners, such as MBD1 in chromatin may create a reservoir of repair proteins to be released in response to alkylation damage [22]. Murine embryonic stem (ES) cells deficient in AAG have been constructed by gene knockout technology [24]. These cells are hypersensitive to killing by MMS and other simple alkylating agents, and are susceptible to MMS-induced sister chromatid exchange. It was concluded that AAG “plays a pivotal role in preventing alkylation-induced chromosome damage and cytotoxicity” in ES cells [24]. Surprisingly, subsequent studies on knockout mice deficient in AAG showed a phenotype of only marginal alkylation sensitivity with no increased cancer frequency or reduced life span [25], [26]. Fibroblasts and bone marrow cells from such mice exhibit resistance to MMS-induced cytotoxicity although no other 3meA-DNA glycosylase has been detected [27]. Possibly, this resistance is due to a translesion DNA polymerase. It would be interesting to know whether any such polymerase is expressed in differentiated cells and tissues but not in ES cells.
    O6-Methylguanine is a miscoding base in DNA generated by exposure to SN1 alkylating agents, such as methylnitrosourea. It is repaired by direct damage reversal with transfer of the methyl group to a specific cysteine residue in the repair enzyme itself (Fig. 2). In vivo, the methylated MGMT protein is degraded by the ubiquitin pathway rather than being reactivated by demethylation. This is probably because the strength of the methyl-cysteine covalent bond makes demethylation of the protein too difficult. The unusual suicidal mechanism of this repair function and the possibility that active depletion of MGMT might sensitise cells to alkylating agents has been reviewed frequently in recent years, e.g., [28], [29], [30] so will not be described in detail here. The 3D structure of human MGMT has been established and shows that an O6meG residue in DNA is flipped out to access the methyl receptor cysteine residue [31]. The 22kDa enzyme comprises two subdomains that are inactive when separated but regain activity when mixed together [32]. Many organisms including E. coli and S. pombe produce an alkyltransferase-like protein (ATL) with a Trp instead of a Cys residue in the position equivalent to the MGMT active site. These ATL proteins bind specifically to O6meG-containing double-stranded oligonucleotides but cannot demethylate them; their physiological role is currently unknown [33].