The biochemical basis of 5-bromouracil- and 2-aminopurine-induced mutagenesis.

We describe in vitro measurements of heteroduplex base mispaired intermediates involving 5-bromouracil and 2-aminopurine in A X T----G X C and G X C----A X T transition mutation pathways. For the case of 2-aminopurine, 2-aminopurine X cytosine mispairs are formed at a much higher frequency than adenine X cytosine mispairs in either transition pathway. For the case of 5-bromouracil, at least a 40-fold increase in 5-bromouracil X guanine mispairs are observed over thymine X guanine mispairs but only in the G X C----A X T pathway. In the A X T----G X C pathway, mispairs involving 5-bromouracil are formed 2.5-fold more frequently to those involving thymine suggesting perhaps that 5-bromouracil may exhibit substantially different base-pairing behavior depending on whether it is present as a template base or as a deoxyribonucleosides triphosphate substrate. The effect of the base analogs on dNTP pool size perturbations is discussed. A measurement of dNTP pools in 2-aminopurine mutagenized bacteriophage T4-infected cells is presented. An approximate eight-fold expansion in common dNTP pools is observed in a ts L141 antimutator genetic background compared to wild type T4 43+ and ts L56 mutator backgrounds. The effects of distorted dNTP pools on mutagenesis will be considered.

On the molecular basis of transition mutations. Frequency of forming 2-aminopurine-cytosine base mispairs in the G X C----A X T mutational pathway by T4 DNA polymerase in vitro.

An in vitro model system including wild-type T4 DNA polymerase, the mutagenic nucleotide analogue 2-aminopurine deoxyribonucleoside triphosphate, and poly[d(A,C)] X oligo(dT) poly(dC) X oligo(dG) template-primers is used to measure the frequency of 2-aminopurine X cytosine base mispairs formed in the G X C----A X T mutational pathway. Incorporation and turnover of the analogue into DNA is dependent on the presence of cytosine on the template strand and is reduced significantly in the presence of dGTP. 2-Aminopurine X cytosine mispairs are observed to occur at a 2-3 order of magnitude greater frequency than adenine X cytosine mispairs. The frequency of inserting 2-aminopurine deoxyribonucleoside monophosphate in place of dGMP opposite template cytosine sites is about 3-6% when either strong or weak base-stacking partners are present on the primer strand. However, enzymatic proofreading of the mispair strongly depends on base-stacking partners. Greater than 85% of misinserted 2-aminopurine deoxynucleotides are excised whenever the mispairs are formed next to 5'-primer thymine sites. A 5-fold reduction in proofreading frequency occurs when the mispair is formed with 2-aminopurine deoxynucleoside monophosphate stacked adjacent to a 5'-primer guanine. The frequency of 2-aminopurine X cytosine base mispair formation in the G X C----A X T pathway is similar to that found previously in the A X T----G X C pathway (Watanabe, S. M., and Goodman, M.F. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2864-2868). We propose a criterion for base selection by DNA polymerase to account for the unexpected similarity in base mispairing rates in the two transition pathways.

Analysis of alkylated sites at N-3 and N-7 positions of purines as an indicator for chemical carcinogens.

This study reports a rapid assay to distinguish depurination from other forms of alkaline-labile lesions induced in DNA by alkylating agents. Covalently closed circular duplex PM2 DNA was treated with various alkylating agents such as N-methyl-N-nitrosourea, dimethyl sulfate, methyl methanesulfonate, N-ethyl-N-nitrosourea, diethyl sulfate, and ethyl methanesulfonate at pH 6.5. Apurinic sites and subsequent strand breaks were introduced by the hydrolysis of the alkylated purines under nondenaturing conditions by heating alkylated DNA at 70 degrees for 1.5 hr with 0.05 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid:KOH (pH 7.4), 0.1 M KCl, 0.01 M MgCl2, 0.0005 M ethylenediaminetetraacetate, 0.05 M glycine, and 0.01 M putrescine. The number of strand breaks produced, representing the alkylated sites at N-3 and N-7 positions of purines, were quantitated by electrophoresis in 1% neutral agarose slab gels. These results were compared with previously reported carcinogenic and mutagenic effects of these compounds, and a correlation between the apurinic sites, the total alkylated sites, and the biological effect of the alkylating agent was determined.