CC-1065 and the pyrrolo(1,4)benzodiazepines - anthramycin, sibromycin and tomamycin - are potent antitumor antibiotics. The cytotoxicity of these drugs appears to derive from drug-DNA interactions. Each drug shows a different sequence specificity, but in each case the drug-DNA adducts lie snugly cradled within the minor groove of the DNA helix in a nondistortive fashion. Consequently, the melting temperature of the DNA is increased, as well as its resistance to S1 nuclease. DNA damage induced by UV and polycyclic hydrocarbons has the opposite effect. The objectives of this proposal are: 1) to determine the role of these DNA adducts in genotoxicity and mutagenicity, and 2) to investigate how the sequence specificity of drug binding and drug-induced DNA conformational changes affect the DNA repair processes. In order to study the genotoxicity of drug-DNA adducts per se and to investigate the particular gene product involved in their repair, the transfectivity of drug modified PhiX174 RF DNA in E. coli mutants (uvrA, uvrB, uvrC, uvrD, gyrB, or topA) will be assessed. We have already found that uvrA and uvrB mutants are more sensitive than wild-type cells to anthramycin-DNA adducts, while uvrC mutants are more resistant. We will explore the biochemical basis for these findings by examining 1) how these drug-DNA adducts are processed in E. coli cells, 2) the effect of UVRD protein on the reaction between the UVRABC enzymes and DNA adducts, and 3) whether topoisomerases which are involved in modulating DNA topological changes play a role in repair of these drug-DNA adducts. A second aspect of our work is to investigate the effect of these DNA adducts on the fidelity of DNA replication. To allow the detection of a boader spectrum of mutations, an M13mp10 lacZ forward mutation system will be used. By sequencing the mutant phage DNA, we should be able to detect base changes that result from these adducts. In order to obtain unambiguous results, we propose to use synthetic DNA sequences which contain a single site for drug binding. These sequences will be used to determine 1) the mode of reaction between UVRABC + UVRD enzymes and different DNA adducts, and 2) the effect of the adduct and its surrounding sequences on the fidelity of DNA replication. The results of these studies should provide insight into the recognition and repair of DNA damage, and may provide useful information for designing drugs which retain antitumor potency but are themselves non-carcinogenic.
