A human DNA repair activity specific for O4-ethylthymine: identification and partial characterization

Abstract

An O4-ethylthymine-specific antibody and immunoslot blot assay were used to identify an enzymatic activity in human tissue and cell extracts, specific for removing O4-ethylthymine from ethylated DNA in vitro. The assay allowed the quantitation of activity in the range of 2 fmol O4-ethylthymine removed per mg cell-free protein extract. The specific activities in human brain, kidney and liver tissue extracts ranged from 5.2 to 26.0 with mean levels of 12.8, 9.9 and 12.3 fmol/mg protein respectively. The activity in extracts prepared from cultured kidney and liver epithelial cells ranged from 8.4 to 16.0 fmol/mg protein, exhibiting mean levels of 10.3 and 13.5 fmol/mg protein respectively. The similar levels of repairing O4-ethylthymine in human liver, kidney and brain contrast the organ-specific activity of the O6-alkylguanine DNA-methyltransferase. The repair activity for O4-ethylthymine was not inhibited by preincubation of the extracts with either O4-methylthymine or O6-methylguanine, indicating that the loss of O4-ethylthymine was not mediated by an alkyltransferase. However, there was no detectable activity in extracts prepared from the GM006 mer- cell line lacking O6-alkylguanine DNA-methyltransferase. These data suggest that the activity for repairing O4-ethylthymine may be due to a protein distinct from the O6-alkylguanine DNA-methyltransferase. The low, but significant, level of repair activity specific for O4-ethylthymine identified in human tissue and cell extracts is consistent with the slow, but active, repair of this adduct in vivo.