Abstract
Like the E. coli homologs, both of the known thymine glycol (Tg)-DNA glycosylases in mouse cell, mNTH1 and mNEIL1, possess bifunctional activities of Tg-DNA glycosylase and AP lyase. We have reported a novel monofunctional Tg-DNA glycosylase activity in nuclear extract of mouse organs. We purified the activity from nuclei in mouse organs with ammonium sulfate precipitation, hydrophobic, hydroxyapatite and ion exchange column chromatography, but could not completely separate the Tg-DNA glycosylase activity from minor AP lyase activity. When both activities were compared after different reaction time using the partially purified fraction from mouse stomach with Ceramic Hydroxyapatite Column (BIO-RAD), the reaction rate of Tg-DNA glycosylase activity was 6-times higher than that of AP lyase activity, suggesting either the small contamination of lyase or the association of the minor activity with major Tg-DNA glycosylase activity. Thus, we performed electrophoretic mobility shift assay of partially purified fractions from mouse lung with sodium borohydrate. The intensity of the protein-nucleotides band with a molecular mass of about 41-44 kDa correlated with the levels of Tg-DNA glycosylase activity and minor AP lyase activity, indicating the presence of about 35- 38 kDa protein which bound Tg-DNA substrate to form a covalent bond by reduction.
