Abstrakt

Numerous antitumor and carcinogenic compounds are able to modify DNA by forming covalent bonds with its constituents, while some anticarcinogenic compounds are known to prevent such a modification. All these processes are of vital biological import_ance, though deeper inside into factors influencing formation of DNA adducts is difficult due to the low level of their occurrence. 32P-Postlabelling approach ensures very sensitive detection of DNA binding induced by a variety of genotoxins, however, the employment of this technique to mechanistic studies is too expensive, time consuming and sort of restricted by the risk associated with the exposure to radiochemicals to become widely applied. In our research, we were trying to workout a simple approach that would enable optimisation of conditions of DNA binding or its prevention in cell-free system as a preliminary step before 32P-postlabeling experiments. As a result, the DNA restriction analysis method to detect DNA covalent modification has been developed. This method uses a DNA amplicon that is 695 bp long and contains two restriction sites: one consisting of only GC base pairs recognized by restriction endonuclease MspI or HpaII, methyl-insensitive and methyl sensitive, respectively, and the other consisting of only AT base pairs recognized by the enzyme Tru1I. The covalent modification of the restriction sites abolishes their recognition and thus cleavage by the restriction enzymes. By incubating the DNA amplicon with increasing concentrations of the studied compound(s) in the appropriate activating system, the formation of DNA adducts is induced. Then, the modified amplicon is submitted to digestion by the mentioned endonucleaues and the DNA fragments are separated by polyacrylamide gel electrophoresis. The inhibition of cleavage indicates the occurrence of covalent modification of the restriction site(s) simultaneously pointing at the kind of base pairs involved in DNA adduct formation. The examples will be given demonstrating the application of the described technique to detect DNA modifications, including oxidation of nucleobases, by a number of genotoxic agents. Also, the follow up of kinetics of such reactions and identification of metabolic stages of activation involved in formation of DNA reactive species will be illustrated.

Autorzy (5)