Demethylation of 3-methylthymine in DNA by bacterial and human DNA dioxygenases.
Rare DNA lesions that are chemically stable and refractory to repair may add disproportionately to the accumulation of mutations in long lived cells. 3-Methylthymine is a minor lesion that is induced by DNA-methylating agents and for which no repair process has been described previously. Here we demonstrate that this lesion can be directly demethylated in vitro by bacterial and human DNA dioxygenases. The Escherichia coli AlkB and human ABH3 proteins repaired 3-methylthymine in both single-stranded and double-stranded polydeoxynucleotides, whereas the human ABH2 protein preferred a duplex substrate. Thus, the known substrates of these enzymes now include 3-methylthymine in DNA, as well as 1-methyladenine and 3-methylcytosine, which all have structurally similar sites of alkylation. Repair of 3-methylthymine by AlkB and ABH3 was optimal at pH 6, but inefficient. At physiological pH, 3-methylthymine, which is a minor methylated lesion, was more slowly repaired than the major lesion generated in single-stranded DNA, 3-methylcytosine. Our data suggest that 3-methylthymine residues in DNA will be repaired inefficiently in vivo and therefore may occur at a low steady-state level, but the residues should not gradually accumulate to high levels in long lived cells.
Koivisto
Pertti
P
Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, United Kingdom.
Robins
Peter
P
Lindahl
Tomas
T
Sedgwick
Barbara
B
D000225:Adenine / Q000031:analogs & derivatives* / Q000737:chemistry; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003596:Cytosine / Q000031:analogs & derivatives* / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D019175:DNA Methylation; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006899:Mixed Function Oxygenases / Q000737:chemistry*; D008956:Models, Chemical; D011108:Polymers / Q000737:chemistry; D013941:Thymine / Q000031:analogs & derivatives* / Q000737:chemistry*; D013997:Time Factors
D004277:DNA, Single-Stranded; D011108:Polymers; C036386:3-methylcytosine; C008407:1-methyladenine; D003596:Cytosine; D004247:DNA; D006899:Mixed Function Oxygenases; D000225:Adenine; D013941:Thymine; C002347:3-methylthymine