Mutagenesis in vitro by depurination of ΦX174 DNA

Abstract

Depurination, the spontaneous release of purine bases from DNA, occurs by the breakage of the N- glycosylic bond between the purine base and the deoxyribose moiety. The resulting apurinic site is relatively stable, with an in vitro half life of up to several days1. The rate constant of depurination in vitro has been estimated to be 1.8×10−9 per min (ref. 2). This corresponds to the formation of 20,000 apurinic sites per mammalian cell per day. In addition, much of the damage to DNA by chemical carcinogens occurs through covalent binding to the N-3 and N-7 positions on the purine bases3,4, enhancing the rate of depurination of that base by three to four orders of magnitude5–7. The fact that cells have multiple pathways for removing apurinic sites8,9 and even a mechanism for reinserting purines10 suggests that the presence of apurinic sites may be highly detrimental. Apurinic sites in DNA have been postulated as potential mutagenic lesions11, although there has been little experimental support for this concept. We have previously demonstrated that depurination of synthetic polynucleotide templates leads to increased mis-incorporation of non-complementary nucleotides12,13. We now present evidence that a DNA polymerase can copy past apurinic sites on natural, biologically active DNA and further, that this leads to increased mutagenesis.