Quantitative assays for uracil-DNA glycosylase of high sensitivity

Abstract

We have developed a sensitive fluorometric assay using bisulfite deaminated (C----U), covalently-closed circular PM2 DNA as the substrate. We describe a reliable way to prepare this substrate without nicking the PM2 DNA. Methods, which depend on toluenization of the cells, are described for reproducibly and quantitatively assaying uracil-DNA glycosylase. The sensitivity is such that only 200 EL4 mouse thymoma cells or 30,000 Escherichia coli cells are needed for each point in a kinetic assay.