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Removal of purine bases from phi X174 single-stranded DNA leads to increased reversion frequency of amber mutations when this DNA is copied in vitro with purified DNA polymerases. This depurination-induced mutagenesis is observed at three different genetic loci and with several different purified enzymes, including Escherichia coli DNA polymerases I and III, avian myeloblastosis virus DNA polymerase, and eukaryotic DNA polymerases alpha, beta, and gamma. The extent of mutagenesis correlates with the estimated frequency of bypass of the lesion and is greatest with inherently inaccurate DNA polymerases which lack proofreading capacity. With E. coli DNA polymerase I, conditions which diminish proofreading result in a 3-5-fold increase in depurination-induced mutagenesis, suggesting a role for proofreading in determining the frequency of bypass of apurinic sites. The addition of E. coli single-stranded DNA-binding protein to polymerase I catalyzed reactions with depurinated DNA had no effect on the extent of mutagenesis. Analysis of wild-type revertants produced during in vitro DNA synthesis by polymerase I or avian myeloblastosis virus DNA polymerase on depurinated phi X174 amber 3 DNA indicates a preference for insertion of dAMP opposite the putative apurinic site at position 587. These results are discussed in relation both to the mutagenic potential of apurinic sites in higher organisms and to studies on error-prone DNA synthesis.

The role of pyrimidine dimers in mutagenesis by ultraviolet light was examined by measuring the UV-induced reversion of six different bacteriophage M13 amber mutants for which the neighboring DNA sequences are known. The mutational response at amber (TAG) codons preceded by a guanine or adenine (where no pyrimidine dimer can be formed) were compared with those preceded by thymine or cytosine (where dimer formation is possible). Equivalent levels of UV-induced mutagenesis were observed at both kinds of sites. This observation demonstrates that there is no requirement for a pyrimidine dimer directly at the site of UV-induced mutation in this single-stranded DNA phage. UV irradiation of the phage was also performed in the presence of Ag+ ions, which specifically sensitize the DNA to dimer formation. The two methods of irradiation, when compared at equal survival levels (and presumably equal dimer frequencies), produced equivalent frequencies of reversion of the amber phage. We believe these results indicate that while the presence of pyrimidine dimers may be a prerequisite for UV mutagenesis, the actual mutagenic event can occur at a site some distance removed from a dimer.

The possible consequences of depurination for both spontaneous and induced mutagenesis were investigated using in vitro and in vivo assays. Depurination of synthetic polynucleotide templates such as poly[d(A-T))] or poly[d(G-C)] leads to increased misincorporation of noncomplementary nucleotides when these templates are copied by prokaryotic and eukaryotic DNA polymerases.

It is well known that the high fidelity of DNA replication in eucaryotes results in part from the accuracy by which DNA polymerases copy the base sequences in DNA. It is also well known that the reactions of many mutagens and carcinogens with DNA result in the formation of bulky adducts. Experimental evidence indicates that these bulky adducts block DNA synthesis in vitro and in vivo.1 A paradox in mutagenesis is how DNA polymerases, with their stringent requirement for correct base pairing, can accommodate bulky adducts to yield mutations in nascent DNA. One possibility is that the adducts responsible for mutagenesis are labile and form non-bulky intermediary products that are mutagenic. We have proposed that apurinic sites are mutagenic intermediates2 and we have subsequently analyzed their mutagenic potential in vivo and in vitro.