Direct removal of alkylation damage from DNA by AlkB and related DNA dioxygenases.
The cytotoxic alkylation lesions 1-methyladenine (1-alkyladenine) and 3-methylcytosine are removed efficiently from DNA by direct damage reversal, catalyzed by the Escherichia coli AlkB protein and its human homologs ABH2 and ABH3. The enzymes act by oxidative demethylation, employing Fe(II) and alpha-ketoglutarate as cofactors, and release the methyl moiety as formaldehyde. The isolation of these enzymes from overproducing cells is described, as well as the preparation of radioactively labeled substrates and procedures for enzyme assays. Functionality in vivo is examined by complementation of the low survival of alkylated single-stranded DNA bacteriophage in an E. coli alkB mutant.
Sedgwick
Barbara
B
London Research Institute, Cancer Research United Kingdom, Clare Hall Laboratories, South Mimms, Hertfordshire.
Robins
Peter
P
Lindahl
Tomas
T
D000478:Alkylation; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004249:DNA Damage; D019175:DNA Methylation; D004260:DNA Repair; D049308:Dioxygenases / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015394:Molecular Structure; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D004247:DNA; D006899:Mixed Function Oxygenases; D049308:Dioxygenases; C466427:AlkB protein, E coli