Except for S adenosylmethionine SAM Fig sources of endogenou
Except for S-adenosylmethionine (SAM, Fig. 1), sources of endogenous DNA alkylation are not well defined. Other possible sources include nitroso compounds related to the well known mutagen methylnitrosourea which are generated in vitro by nitrosation of cellular amines including amino acids, proteins and polyamines .
Endogenous DNA alkylation by SAM SAM is the donor of methyl groups in the majority of in vivo enzymatic methylation events involving a wide variety of acceptor molecules. The high transfer potential of SAM however means that it will also spontaneously methylate cellular nucleic acids and proteins at a low but significant rate. SAM acts by an SN2 (bimolecular nucleophilic substitution) mechanism and yields the same products on alkylation of DNA as the experimental alkylating agent, methyl methanesulfonate (MMS). However, the reactivity of SAM is 2000-fold weaker than MMS . SAM is present in various tissues at a concentration of 25 to 50μM . The products detected on treatment of double-stranded DNA with SAM are 7-methylguanine and 3-methyladenine (3meA). Whilst 7-methylguanine is relatively innocuous, 3meA has a strong toxic effect. The methyl group of 3meA does not perturb interaction with the complementary DNA strand, rather it protrudes into the minor groove of the double helix which is normally free of methyl groups (the methyl groups of thymine and 5-methylcytosine are in the major groove). Here, 3meA efficiently blocks RNA- and most DNA polymerases resulting in a strong cytotoxic but feeble mutagenic effect. In these early studies, SAM-induced methylation of single-stranded DNA was not examined. However, other SN2 methylating agents generate substantial amounts of 1-methyladenine (1meA) and 3-methylcytosine (3meC) in single-stranded DNA , . These lesions are unable to UNC669 pair and block DNA replication resulting in a strong toxic but weak mutagenic effect similar to 3meA. The low mutagenicity observed may be due to translesion synthesis by non-replicative DNA polymerases.
Mammalian 3-alkyladenine-DNA glycosylase (MAG or AAG) Whilst some bacteria contain two or more distinct DNA glycosylases that catalyze the excision of 3meA from DNA, mammalian cells have only one (MAG, also called AAG for 3-alkyladenine-DNA glycosylase) (Fig. 2). Interestingly, mammalian AAG shows regional similarities to Bacillus subtilis and Arabidopsis thaliana 3meA-DNA glycosylases but is entirely different from E. coli, S. cerevisiae and S. pombe enzymes. In contrast, E.coli, yeast and human uracil-DNA glycosylases (UNG) show strong sequence homology . This apparent difference in conservation between the UNG enzymes and the 3-methyladenine-DNA glycosylases can be explained by considering the relative stability of the glycosyl bonds of their substrates. A dUMP residue in DNA is as stable as the four common nucleotides. An efficient and highly specialized enzyme is therefore required to release uracil by promoting cleavage of the uracil-deoxyribose bond by more than 107-fold. By contrast, a 3meA-deoxyribose bond is intrinsically unstable with a half-life of about 26h at 37°C, pH 7 . As a result, a glycosylase that can detect and flip out this residue in DNA and then promote cleavage of the glycosyl bond by a mere 1000-fold could succeed in excising 3meA; apparently this goal can be achieved by glycosylases using several different strategies. Resolution of the three-dimensional structure of human AAG revealed a unique fold and provided substantial information on the base excision mechanism . AAG binds and slightly widens the minor groove of DNA. The enzyme then inserts an aromatic residue into the helical stack as a probe for the altered base. This probably occurs during rapid sliding of AAG along the DNA by diffusion as was recently established for the Fpg/MutM glycosylase and its eukaryotic counterpart Ogg1 , . After recognition of a site of damage, AAG may trigger flipping-out of the altered deoxynucleoside sandwiching it between two aromatic amino acid residues in the active site of the enzyme. The positive charge of the 3meA residue will encourage these stacking interactions, whilst a strategically bound water molecule may then promote hydrolysis of the glycosyl bond , .