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  • Expression of MGMT in human cells can be switched


    Expression of MGMT in human gtpase inhibitor can be switched off by enzymatic methylation of a cytosine residue in the MGMT promoter sequence. In this regard, both MGMT and the mismatch repair function MLH1 belong to a subset of DNA repair genes that are subjected to epigenetic control. In consequence, about one-third of human tumor cell lines do not express MGMT [34], [35].
    Induction of repair of alkylated DNA in bacteria The Ada protein of E.coli repairs DNA and regulates the bacterial adaptive response to methylation damage [36]. This protein's C-terminal domain directly demethylates O6meG residues in DNA, a repair function similar to that of the mammalian MGMT protein. The N-terminal domain regulates expression of its own gene and that of three other genes whose products influence alkylation resistance. Ada was the first example of a transcription factor activated by post-translational modification [37]. Self-modification occurs by transfer of a methyl group from an innocuous methyl phosphotriester DNA lesion, the Sp diastereoisomer, to a Cys residue in the N-terminal domain. The modified protein has a greatly increased binding affinity for the promoters of the ada-alkB operon, and the alkA and aidB genes. This Ada N-terminal domain function has no direct counterpart in eukaryotes. Recently, the molecular details of the Ada transcriptional switch have been determined by structural investigations using NMR, X-ray crystallography, and mass spectrometry [38], [39], [40]. Four invariant Cys residues (Cys-38, -42, -69, and -72) bind tightly a single zinc ion which is a unique requirement for methyl transfer. The protruding methyl group of the Sp phosphotriester stereoisomer is transferred to Cys-38. The Cys-42, -69, and -72 sites are protected from such methylation by hydrogen bonding interactions. Methylation of the Cys-38 residue does not cause a structural rearrangement of the N-terminal domain and the overall folding of the Ada protein remains unchanged. Instead, methylation of Cys-38 alleviates a repulsive DNA-protein charge interaction, so that Ada activation is controlled by a simple methylation-dependent electrostatic switch [39].
    The DNA dioxygenase AlkB and its human homologs ABH2 and ABH3
    Crystal structures of AlkB and ABH3 The X-ray crystal structure of the E.coli AlkB protein in enzyme–substrate complexes was recently resolved, and revealed details of the mechanism of DNA demethylation [52]. The N-terminal 11 amino acid residues of AlkB not required for enzyme activity were deleted to permit crystal formation. Crystals were formed under anaerobic conditions with bound cofactors Fe2+ and 2OG, and the methylated trinucleotide substrate T(1meA)T [52]. In aerobic conditions, this trinucleotide is repaired by AlkB [51], [52]. The AlkB structure consists of three well-defined regions; a catalytic core in the carboxy-terminal domain, a unique nucleotide-recognition lid and an N-terminal extension (Fig. 4A). The organisation of the catalytic core matches that of other members of the superfamily of 2OG-Fe2+-dioxygenases [75]. It is comprised of two β-sheets arranged in a rigid jelly-roll topology along with a conserved Hx1DxNH motif that coordinates Fe2+ and two conserved arginine residues found at the beginning and end of the last β-strand which form stabilizing salt bridges with the carboxylates of 2OG [44] (see Fig. 4A and B). The nucleotide-recognition lid consists of a flexible β-sheet that closes over the DNA substrate locking the methylated adenine base over the catalytic-site. The intrinsic flexibility of the DNA-binding region has been confirmed by NMR studies (T. Shivarattan and S. Matthews, in preparation). Flexibility within the lid region is reduced upon substrate-binding [52]. Motility in the absence of a substrate may explain why AlkB can repair a diverse range of alkylated substrates [51], [56], [57]. Oxidation of bulky alkyl groups will require re-orientation of the base and movement of the polynucleotide backbone to align the target group with the catalytic centre. The binding geometry of larger lesions must be less than optimal for catalysis because methylated bases are better AlkB substrates but further X-ray structures will be required to explain such substrate preferences. An additional 3D structure of interest would be a complex of AlkB with a double-stranded rather than a single-stranded oligonucleotide containing a 1meA residue.