Quantitative assays for uracil-DNA glycosylase of high sensitivity.
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.
Morgan
A R
AR
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Chlebek
J
J
D000818:Animals; D045647:DNA Glycosylases; D004260:DNA Repair; D004270:DNA, Circular; D003641:Deamination; D051379:Mice; D009699:N-Glycosyl Hydrolases / Q000032:analysis*; D013945:Thymoma / Q000201:enzymology; D013953:Thymus Neoplasms / Q000201:enzymology; D014407:Tumor Cells, Cultured / Q000201:enzymology; D051981:Uracil-DNA Glycosidase
D004270:DNA, Circular; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase