Abstract
It has been accepted that most of oxidized pyrimidines formed in mouse genome were exclusively removed by two bifunctional DNA glycosylases, mNTH1 and mNEIL1. Since EGFP-tagged mNTH1, unlike the human homolog, was localized mostly in mitochondria, it was considered that mNEIL1 had a major contribution toward the repair of oxidized pyrimidines in mouse nuclei. Recently, a monofunctional DNA glycosylase, SMUG1, was revealed in nuclei of mammalian cells. The DNA glycosylase removes a group of oxidized pyrimidines, such as 5-hydroxyuracil and 5-formyluracil (fU). Since many monofunctional DNA glycosylases remove a variety of altered bases in mammalian cells, the presence of SMUG1 seems to be reasonable. However, SMUG1 does not remove thymine glycol (TG), 5,6-dihydrothimine(DHT), 5-formylcytosine (fC), and no monofunctional DNA glycosylase against these altered bases has been discovered so far. We found a novel monofunctional TG-DNA glycosylase (TGG) activity in nuclei from several mouse organs. We prepared a nuclear extract without most of mNTH1 and mNEIL1 from mouse organs by a modified high-salt extraction method. We further separated the monofunctional TGG activity from most of apurinic/apyrimidinic (AP) lyase activities with column chromatography. The monofunctional TGG activity within the fraction was determined using radiolabeled oligonucleotide substrates containing TG or AP site. The monofunctional TGG activity removed not only fU, but TG and DHT, which are not removed by SMUG1. Under our reaction condition, an activity against fC was not detected.
