Effects of adduct formation on the biological activity of single- and double-stranded øX174 DNA, modified by N-acetoxy-N-acetyl-2-aminofluorene.

In order to establish a good quantitative relationship between the number of acetylaminofluorene adducts and the extent of inactivation of DNA, single-stranded (ss) øX174 DNA and øX174 RF DNA were modified to various extents with 3H-labelled N-acetoxy-N-acetyl-2-aminofluorene (N-AcO-AAF) and subsequently transfected to Escherichia coli spheroplasts having different repair capabilities. Exponential survival curves were obtained. In the case of ssDNA about one adduct per molecule appears to be lethal. On the other hand only 1 out of 10.2 adducts is found to inactivate RF DNA if tested on wild-type E. coli. However, when assayed on strains deficient in excision repair 1 out of 2.3 adducts leads to inactivation of RF DNA. RecA-dependent postreplication repair only has little influence on these figures. Product analysis of the modified DNAs shows that in RF DNA at least 76% of the interaction products is N-(deoxyguanosin-8-yl)-N-acetyl-2-aminofluorene (dGuo-C8-AAF) and at least 6% and at most 12% is 3-(deoxyguanosin-N2-yl)-N-acetyl-2-aminofluorene (dGuo-N2-AAF). In ssDNA only dGuo-C8-AAF is formed. No apurinic sites could be detected in the modified DNAs. From these results it can be concluded that in RF DNA most of the dGuo-C8-AAF is removed by excision repair. The remaining damage, consisting probably both of dGuo-N2-AAF and unexcised dGuo-C8-AAF, inactivates RF DNA. Inactivation can be explained by a model which shows that only damage in the minus strand of RF DNA inhibits replication and/or transcription.