A 55-KDA PROTEIN ISOLATED FROM HUMAN-CELLS SHOWS DNA GLYCOSYLASE ACTIVITY TOWARD 3,N-4-ETHENOCYTOSINE AND THE G T MISMATCH/

Etheno adducts in DNA arise from multiple endogenous and exogenous sou rces. Of these adducts we have reported that, 1,N-6-ethenoadenine (eps ilon A) and 3,N-4-ethenocytosine (epsilon C) are removed from DNA by t wo separate DNA glycosylases. We later confirmed these results by usin g a gene knockout mouse lacking alkylpurine-DNA-N-glycosylase, which e xcises epsilon A. The present work is directed toward identifying and purifying the human glycosylase activity releasing epsilon C. HeLa cel ls were subjected to multiple steps of column chromatography, includin g two epsilon C-DNA affinity columns, which resulted in >1,000-fold pu rification. Isolation and renaturation of the protein from SDS/polyacr ylamide gel showed that the epsilon C activity resides in a 55-kDa pol ypeptide, This apparent molecular mass is approximately the same as re ported for the human G/T mismatch thymine-DNA glycosylase, This latter activity copurified to the final column step and was present in the i solated protein band having epsilon C-DNA glycosylase activity, In add ition, oligonucleotides containing epsilon C.G or G/T(U), could compet e for epsilon C protein binding, further indicating that the epsilon C -DNA glycosylase is specific for both types of substrates in recogniti on. The same substrate specificity for epsilon C also was observed in a recombinant G/T mismatch DNA glycosylase from the thermophilic bacte rium, Methanobacterium thermoautotrophicum THF.