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The mutagenic potential of apurinic sites in vivo has been studied by transfection of depurinated phi X174 DNA containing amber mutations into SOS-induced Escherichia coli spheroplasts. Mutagenicity is abolished by treatment of the depurinated DNA with an apurinic endonuclease from Hela cells, establishing the apurinic site as the mutagenic lesion. The frequency of copying apurinic sites in vitro was analyzed by measuring the extent of DNA synthesis using E. coli DNA polymerase I and avian myeloblastosis DNA polymerase. The inhibition of DNA synthesis by apurinic sites was less with avian myeloblastosis DNA polymerase, suggesting that this error-prone enzyme copies apurinic sites with greater frequency. Consistent with this conclusion is the observation that, upon transfection into (normal) spheroplasts, the reversion frequency of depurinated phi X174 am3 DNA copied with avian myeloblastosis virus DNA polymerase is much greater than that of the same DNA copied with E. coli DNA polymerase I. Sequence analysis of the DNA of 33 revertant phage produced by depurination indicates a preference for incorporation of deoxyadenosine opposite putative apurinic sites. The combined results indicate that mutagenesis resulting from apurinic sites is associated with bypass of these noncoding lesions during DNA synthesis.

When bacteriophage φX174 am3 DNA depurinated in vitro is transferted into E. coli spherplasts prepared from bacteria previously exposed to UV light, a strong mutagenic response is observed. This mutagenic response does not occur in spheroplasts derived from pre-irradiated bacteria carrying defective recF, recF or umuC genes. These findings indicate that mutagenesis at apurinic sites is an SOS-dependent process. The mutagenic response is not dependent on the multiplicity of transfection. This suggests that mutagenesis is not mediated by recombination.

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.

Introduction of apurinic sites into phi X174 am3 DNA leads to loss of biological activity when measured in a transfection assay. For single-stranded DNA, approximately one apurinic site constitutes a lethal hit; for double-stranded (RFI) DNA, approximately 3.5 hits per strand are lethal. When the reversion frequency of am3 DNA is measured, no increase due to depurination is observed above the background level. However, a large increase in reversion frequency is observed when the same DNA is assayed by using spheroplasts derived from bacteria previously exposed to UV light. The results suggest that apurinic sites are impediments to a replicating DNA polymerase; however, nucleotides can be incorporated opposite these sites under SOS-induced conditions. We estimate the frequency of mutagenesis per apurinic site to be less than 1 in 1400 in normal spheroplasts and 1 in 100 in SOS-induced spheroplasts.

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.

Errors in DNA replication must be very infrequent, so as not to alter essential genetic information; yet they must occur rarely so as to permit species divergence through mutations. On the basis of rates of appearance of spontaneous mutations, 10−7 to 10−11, DNA replication in vivo is assumed to be highly accurate (1). If one considers the multiplicity of genes demonstrated to affect mutation rates, it seems reasonable that this phenomenal accuracy is achieved through a multi-step process. We have focused on the fidelity by which DNA polymerases copy synthetic polynucleotides and natural DNA and the effects of mutagenic compounds on this accuracy. The underlying hypothesis is that infidelity by DNA polymerase, either on normal or damaged DNA, is a major determinant in mutagenesis.

The fidelity with which E. coli DNA polymerase I (Pol 1) copies an amber mutation in ФX174 DNA has been determined. The frequency of mis-incorporation by the polymerase was calculated from the observed change in reversion frequency of progeny phage in a transfection assay of the copied DNA. The calculated error rate of Pol I in Mg 2+ -activated reactions in the presence of equi-molar concentrations of the four deoxynucleotide sub-strates is 1/680,000 for an A:C mis-pair and less than 1/6,100,000 for an A:A mis-pair at position 587 in ФX174 DNA. In initial experiments, an even greater accu-racy is exhibited by E. coli Pol III holoenzyme. Furthermore, E. coli single-stranded binding protein in-creases the accuracy of DNA polymerases by as much as 20-fold, by a mechanism that may involve immobilization of the template. From the observed reversion frequency of the phage, we have calculated that the mutation rate in vivo at the am3 codon is approximately 1 in 10 8 . Thus, fidelity observed in vitro with only purified DNA polymerases and single-stranded binding proteins approaches that observed in vivo .

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.