A 55-kDa protein isolated from human cells shows DNA glycosylase activity toward 3,N4-ethenocytosine and the G/T mismatch.
Etheno adducts in DNA arise from multiple endogenous and exogenous sources. Of these adducts we have reported that, 1,N6-ethenoadenine (epsilonA) and 3,N4-ethenocytosine (epsilonC) are removed from DNA by two separate DNA glycosylases. We later confirmed these results by using a gene knockout mouse lacking alkylpurine-DNA-N-glycosylase, which excises epsilonA. The present work is directed toward identifying and purifying the human glycosylase activity releasing epsilonC. HeLa cells were subjected to multiple steps of column chromatography, including two epsilonC-DNA affinity columns, which resulted in >1,000-fold purification. Isolation and renaturation of the protein from SDS/polyacrylamide gel showed that the epsilonC activity resides in a 55-kDa polypeptide. This apparent molecular mass is approximately the same as reported for the human G/T mismatch thymine-DNA glycosylase. This latter activity copurified to the final column step and was present in the isolated protein band having epsilonC-DNA glycosylase activity. In addition, oligonucleotides containing epsilonC.G or G/T(U), could compete for epsilonC protein binding, further indicating that the epsilonC-DNA glycosylase is specific for both types of substrates in recognition. The same substrate specificity for epsilonC also was observed in a recombinant G/T mismatch DNA glycosylase from the thermophilic bacterium, Methanobacterium thermoautotrophicum THF.
Hang
B
B
Donner Laboratory, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720, USA.
Medina
M
M
Fraenkel-Conrat
H
H
Singer
B
B
D000818:Animals; D001483:Base Sequence; D003596:Cytosine / Q000031:analogs & derivatives* / Q000378:metabolism; D045647:DNA Glycosylases; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D044502:Thymine DNA Glycosylase
C018289:3,N(4)-ethenocytosine; D003596:Cytosine; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D044502:Thymine DNA Glycosylase; C113921:mismatch-specific thymine uracil-DNA glycosylase