Stereoselective excision of thymine glycol lesions by mammalian cell extracts
2004
Abstract Thymine glycols (Tg) are major pyrimidine oxidation products produced by chemical agents and ionizing radiation. Recent improvements in purification procedures gave us the opportunity to examine the incision of DNA duplexes containing a single (5 S ,6 R )- or (5 R ,6 S )-Tg lesion by mouse NTH1 DNA glycosylase and mammalian cell nuclear extracts. Time course experiments and steady state enzyme kinetics indicated that mNTH1 discriminates between the cis -Tg isomers. In addition, a variety of mammalian cell nuclear extracts showed a similar discrimination between the cis -Tg isomers. Trapping of Schiff base intermediates with sodium borohydride demonstrated that a single protein–DNA complex was formed in the presence of the nuclear extracts. The electrophoretic mobility of trapped complexes formed with both Tg isomers was identical to one another and similar to that of the complex formed with recombinant mNTH1. These results suggest that among all Tg-active DNA glycosylases, NTH1 is the major enzyme in mammalian cell nuclear extracts responsible for incision of duplexes containing cis -Tg isomers.
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