THE BIOCHEMISTRY OF DNA ALKYL PHOSPHOTRIESTERS

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Quantitatively the greatest DNA oxygen lesion produced by a family of 1-alkyl-1-nitroso compound carcinogens, which includes dimethyl and diethylnitrosamine, methyl and ethylnitrosourea and 1-methyl-2-nitro-1-nitrosoguanidine, is at the backbone phosphate groups. The yield is 2-to 5-fold higher than at the 06-position of guanine. DNA oxygen alkylation correlates with carcinogenic potential. It is not known to what degree these alkyl phosphotriesters effect the biochemistry of DNA; I am considering the possibility that they aggravate the binding of proteins which function to insure the proper transmission of genetic information. Using an automatic DNA synthesizer and the Beta-cyanoethyl phosphoramidite chemistry I propose to synthesize DNA polymers of known length and sequence containing any percentage of diastereomerically pure ethyl phosphotriesters. With the view that phosphotriesters could perturb the DNA conformational signals which define protein recognition sites, I will consider the effects of these lesions on DNA helical conformation and conformational stability using helix-coil transition experiments and circular dichroism. I will assess the effects of phosphotriesters on the activity of the single strand binding protein, T4 phage gene 32 protein. Perturbations in the intrinsic binding constant and in the cooperativity parameter will be evaluated by taking advantage of tryptophan fluorescence quenching. The efficiency of E. coli polymerase I on phosphotriester-containing templates will be tested. I will examine the polymerization rate, nucleoside triphosphate turnover and nucleotide incorporation fidelity utilizing standard protocols and HPLC analysis. Finally I will study the effects of phosphotriesters placed at known contact positions of the lac UV5 promoter on the efficiency of E. coli RNA polymerase message initiation using the abortive initiation assay. The promoter fragment will be either synthesized de novo or produced by utilizing some of the methodology developed for oligonucleotide-directed site-specific mutagenesis.