THE BIOCHEMISTRY OF DNA ALKYL PHOSPHOTRIESTERS

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The alkylation of phosphate group oxygens is quantitatively among the major DNA lesions produce in vitro and in vivo by the methylating and ethylating nitroso compounds. The role of these DNA phosphotriesters in the potent carcinogenic and mutagenic activity of these compounds in unknown. DNA phosphotriesters are chemically stable under physiological conditions and persist for relatively long periods in treated cells. There are indications from studies utilizing oligonucleotides that phosphotriesters may influence DNA conformation and conformational stability and also decrease the net rate of DNA polymerization by E. coli polymerase I. We propose experiments which will clearly demonstrate the influence of phosphotriesters on aspects od DNA biochemistry at the polymer level and potentially illustrates the part played by these lesions in the cellular response to alkylating nitroso compound damage. The synthetic DNA homopolymer poly(dT) can be methylated such that better than 98 percent of the alkyl groups are covalently bound to phosphate oxygens; we intend to utilize this modified singlestranded polymer and also the B-form helical duplex it forms with poly(dA) as the substrates in our experiments. We will determine the effects of phosphotriesters on DNA conformation and conformational stability by generating polymer mixing curves, by studying circular dichroism spectra and changes in these spectra with temperature and by observing thermally-induced helix-coli transitions done as a function of monovalent salt concentration. We propose also to study the influences of phosphotriesters in two well characterized DNA-protein interaction systems in which function is expected to be very sensitive to the integrity of the DNA sugar-phosphate backbone. We hope to verify that these lesions decrease the rate of polymerization by E. coli polymerase I and intend to test whether this pertubation is due perhaps to the stalling of polymerization or to changes in processivity. We will also determine whether DNA phosphotriesters induce nucleotide misincorporation. Finally we will consider the effect of phosphotriesters on the cooperative and contiguous binding characteristics of bacteriophage T 4-coded gene 32 protein, the prototype single-stranded DNA binding protein intimately involved in DNA replication, recombination and repair.