DNA glycosylases carry out the first step of base excision repair by removing damaged bases from DNA. The N3-methyladenine (3MeA) DNA glycosylases specialize in alkylation repair and are either constitutively expressed or induced by exposure to alkylating agents. To study the functional and evolutionary significance of constitutive versus inducible expression, we expressed two closely related y...

The acidities of multiple sites in the purine nucleobase adenine (1) and adenine alkyl derivatives 9-ethyladenine (2), 3-methyladenine (3), 1-methyladenine (4), and N,N-dimethyladenine (5) have been investigated for the first time, using computational and experimental methods to provide an understanding of adenine reactivity. We have previously measured two acidic sites on adenine, with the N9 ...

The Escherichia coli AlkB protein, and two human homologs ABH2 and ABH3, directly demethylate 1-methyladenine and 3-methylcytosine in DNA. They couple Fe(II)-dependent oxidative demethylation of these damaged bases to decarboxylation of alpha-ketoglutarate. Here, we have determined the kinetic parameters for AlkB oxidation of 1-methyladenine in poly(dA), short oligodeoxyribonucleotides, nucleot...

The DNA base adenine and four monomethylated adenines were studied in solution at room temperature by femtosecond pump-probe spectroscopy. Transient absorption at visible probe wavelengths was used to directly observe relaxation of the lowest excited singlet state (S(1) state) populated by a UV pump pulse. In H(2)O, transient absorption signals from adenine decay biexponentially with lifetimes ...

Tandem helical repeats have emerged as an important DNA binding architecture. DNA glycosylase AlkD, which excises N3- and N7-alkylated nucleobases, uses repeating helical motifs to bind duplex DNA and to selectively pause at non-Watson-Crick base pairs. Remodeling of the DNA backbone promotes nucleotide flipping of the lesion and the complementary base into the solvent and toward the protein su...

DNA glycosylases preserve genome integrity and define the specificity of the base excision repair pathway for discreet, detrimental modifications, and thus, the mechanisms by which glycosylases locate DNA damage are of particular interest. Bacterial AlkC and AlkD are specific for cationic alkylated nucleobases and have a distinctive HEAT-like repeat (HLR) fold. AlkD uses a unique non-base-flipp...

Two forms of a 6-methyladenine mRNA methyltransferase have been partially purified using a T7 transcript coding for mouse dihydrofolate reductase as an RNA substrate. Both enzyme forms modify internal adenine residues within the RNA substrate. The enzymes were purified 357- and 37-fold respectively from nuclear salt extracts prepared from HeLa cells using DEAE-cellulose and phosphocellulose chr...