Modification of supercoiled DNA with N-cyclohexyl -N'-6-(4- methylmorpholinium) ethyl carbodiimide-p-toluene sulfonate (CMC) produces a drastic decrease in transcription for all superhelical DNAs tested. The inhibition appears to be a one hit event if modification is performed at NaCl concentrations between 100 and 300mM. These results strongly suggest that CMC reacts directly with unpaired bases in promoters and that a single CMC adduct can block RNA polymerase from functioning at some early step in the transcription cycle. We now have definitive methodology to test this hypothesis; CMC adducts located on superhelical templates act as pause or termination sites for the AMV reverse transcriptase using primer extension-DNA sequence methodology. We can precisely map CMC adducts to the nucleotide level. Although only the primer promoter region of ColE1 DNA has been analyzed at this writing, it can be stated unequivocally that CMC adducts are found almost exclusively in this promoter. To develop an understanding of unpairing transitions induced by supercoiling, chemical modification of these sites, and the mechanism(s) of transcriptional inactivation we need to pursue the following research plan: 1. CMC adducts will be mapped throughout the ColE1 DNA molecule. 2. CMC modification will be explored vs. ionic strength, Mg+2 and polyamine concentrations, as well as a function of superhelical density. Topological isomers of ColE1 DNA will be generated for these studies. 3. The effects of CMC adducts on the contact interactions between RNA polymerase and modified promoters will be determined using footprinting and alkylation protection methodology. 4. Abortive initiation kinetics will be used to measure changes in RNA polymerase binding and promoter isomerization constants vs. superhelical density. 5. The possibility of constructing synthetic promoters that can be used to direct CMC to a unique site will be explored with alternative funding and if successful will be applied to this project for definitive testing of inactivation mechanisms. 6. Bromoacetaldehyde modification of supercoiled ColE1 DNA will be examined as an alternative prove of structure-function relationships in supercoiled DNA. It is clearly of considerable interest to understand how chemical damage to DNA can generate cytotoxic, mutageneic and carcinogenic events. We believe that our research strategy will have a major impact in these biomedical areas.
