Rapid sensitive fluorescence assays for DNA endonucleases and DNA glycosylases

Abstract

Using ethidium fluorescence as a probe and the unique topological properties of covalently closed circular (CCC) DNA's, picogram quantities of pancreatic DNase I can be detected. By chemically modifying CCC DNA's without the introduction of nicks, various DNA repair enzymes can be detected even in crude cell extracts. This has previously been done for AP endonucleases from yeast. In this paper we describe the formation of ultraviolet (UV) and bisulfite modified CCC DNA's for the detection of T4 UV endonuclease (a DNA glycosylase + AP endonuclease) and uracil-DNA glycosylases, respectively. These assays should be useful in the isolation of DNA repair mutants.