Determination of target nucleotides involved in 7-methoxy-2-nitro-naphtho[2,1-b]furan (R7000)-DNA adduct formation

The characterization of target nucleotides involved in the binding to DNA of 7-methoxy-2-nitro-naphtho[2,1-b]furan (R7000), a very potent genotoxic nitrofuran derivative, was investigated. Since R7000 undergoes metabolic activation prior to interacting with DNA, plasmids containing AT-rich and GC-rich sequences were devised and treated by R7000 in bacterial cells presenting nitroreductase activity. The nucleotide modifications to these homogeneous fragments that resulted from R7000 treatment were analyzed using the 'postlabeling' method. A preferential binding to the GC segment was demonstrated. Using a modification of the Maxam-Gilbert sequencing technique, it was demonstrated that activated R7000 creates alkali-labile phosphodiester bonds at the positions of guanines. In addition, the analysis of DNA replication-blocking properties of R7000 lesions was performed using avian myeloblastosis virus (AMV) reverse transcriptase as DNA polymerase. The termination of DNA replication occurred preferentially at the sites of guanine residues in the template strand, indicating that one nucleotide was inserted opposite a lesion. All these results indicate that guanine residues are the preferential sites of formation of R7000-DNA adducts.