Use of exonuclease III to determine the site of stable lesions in defined sequences of DNA: the cyclobutane pyrimidine dimer and cis and trans dichlorodiammine platinum II examples

Abstract

A method to detect chemically stable lesions in DNA has been developed using Exonuclease III, a double strand specific nuclease, to digest 5′-end labeled DNA. The products, when analyzed on high resolution DNA sequencing gels, reveal the sites of DNA modification. Cyclobutane pyrimidine dimers induced by UV irradiation can be localized by comparison of the fragments produced by Exonuclease III digestion with fragments obtained after digestion of the DNA with UV specific endonuclease. The experiments demonstrate that Exonuclease III stops one base away from the cyclobutane pyrimidine dimers. Similar experiments with cis- and trans-dichlorodiammine-platinum (II) showed that modifications of DNA by these agents also impede Exonuclease III digestion. In general the same stop sites were found for cis-and trans-platinum adducts. They occur at sites of guanine bases. Additional stop sites were found for cis-platinum at sites of adjacent guanine bases. These results are in agreement with the model that cis-platinum forms intrastrand guanine-guanine dimers, whereas trans-platinum does not.