REVERSE CHEMICAL MUTAGENESIS - IDENTIFICATION OF THE MUTAGENIC LESIONS RESULTING FROM REACTIVE OXYGEN SPECIES-MEDIATED DAMAGE TO DNA

An understanding of the contribution of reactive oxygen species to mut agenesis has been hampered by the vast number of different chemical mo difications they cause in DNA. Even though many of these DNA alteratio ns have been catalogued, the identification of specific lesions that c ause mutations has depended on testing one modification at a time. In this study we present another approach to identify key mutagenic lesio ns from a pool of oxidatively modified nucleotides. dCTP was treated w ith an oxygen radical-generating system containing FeSO4, H2O2, and as corbic acid. The modification products were separated by reverse-phase and anion exchange HPLC and then incorporated by human immunodeficien cy virus reverse transcriptase into a DNA that contains a target gene for scoring for mutations. One of the mutagenic species isolated was i dentified as 5-hydroxy-2'-deoxycytidine. It is incorporated efficientl y into DNA and causes C --> T transitions in Escherichia coli at a fre quency of 2.5%, which is more mutagenic than any previously identified oxidative DNA lesion.