Light-Induced Free-Radical Reactions of Purines and Pyrimidines in Deoxyribonucleic Acid. Effect of Structure and Base Sequence on Reactivity

The multiplicity of damages induced in cellular DNA by treatment with ultraviolet light or with some chemical agents interferes with the assignment of specific types of damages in the DNA to the various biological effects expressed in the treated cells. This problem was approached by developing a series of light-induced alkylation reactions with 2-propanol which led to a single-type chemically defined product of each of the bases in nucleic acids. In the work described here, these reactions were applied to synthetic deoxyribopolynucleotides and to the various forms of øX174 DNA in order to study the effect of sequence and secondary structure on the reactivity of the bases in these reactions. When a series of deoxyribodinucleoside monophosphates and ordered homo- and heterodeoxyribopolynucleotides was used, it was shown that purines having 5'-neighboring pyrimidines are more reactive than those having 5'-neighboring purines. It was thus concluded that the middle purines in the sequences Py-Pw-Py or Py-Pzv-Pu are more reactive than those in the sequences Pu-Pu-Py or Pu-Pm-Pu. The effect of the secondary structure of DNA on the photoalkylation reaction has been studied by comparing the reactivities of the bases in the following 0X174 DNA substrates: single-stranded DNA, whole phage particles, and double-stranded replicative forms (RFI and RFII). Generally, the bases in the single-stranded substrates have been found to react faster than those in the double-stranded forms. The presence of proteins has been found to suppress the rate of alkylation in the phage particles. The bases of the circular superhelical double-stranded form have been found to react faster than those in the relaxed form. The latter result is explained on the basis of the fact that closed circular superhelical DNA contains single-stranded regions which result from unwinding of the DNA duplex. The effect of sequence has been demonstrated by comparing the reactivities of guanines vs. adenines in the single-stranded and RF forms. Our results are compatible with a computer analysis of the frequency of occurrence, in the two forms of the DNA, of triplet sequences with a middle purine. This analysis shows that the abundance of guanine-containing reactive sequences Py-G-Py and Py-G-Pu in single-stranded DNA is higher than that of adenine-containing reactive sequences. In double-stranded DNA, the relative abundance of guanine-containing reactive sequences diminishes. This is consistent with our finding that the reactivity of guanine relative to that of adenine in double-stranded 0X174 DNA is lower than that of the respective bases in the single-stranded form.