Mutational Consequences of Replication of M13mp7L2 Constructs Containing Cis-Opened Benzo[a]pyrene 7,8-Diol 9,10-Epoxide−Deoxyadenosine Adducts

Abstract

The four adducts that arise by cis ring opening of the four optically active benzo[a]pyrene diol epoxides by the exocyclic N6-amino group of deoxyadenosine were incorporated synthetically into each of two different oligonucleotide 16-mers, 5'-TTTXGAGTCTGCTCCC-3' [context I(A)] and 5'-CAGXTTTAGAGTCTGC-3' [context II(A)], at the X position. The eight resultant oligonucleotides were separately ligated into bacteriophage M13mp7L2 and replicated in Escherichia coli that had been SOS-induced, and the progeny were analyzed to evaluate the consequences of replication past these adducts. The presence of these adducts reduced plaque yields substantially. However, the progeny obtained exhibited high frequencies of base substitution mutation ranging from 9 to 68%, depending upon the individual adduct and the sequence context in which it was placed. For most of the adducts, A --> T transversion was the mutation found most frequently in either sequence context, and mutation frequencies in context I(A) were always substantially greater than those in context II(A). In context I(A), adducts with an R configuration at the site of nucleoside attachment were more mutagenic than those with an S configuration. In both sequence contexts that were studied, the cis adduct arising from the (7S,8R)-diol (9S,10R)-epoxide was the most mutagenic adduct. These findings clearly show that individual mutation frequencies are determined by the combined effects of both adduct structure and sequence context.

Crystal structure of a benzo[ a ]pyrene diol epoxide adduct in a ternary complex with a DNA polymerase

“…Synthesis and characterization of the oligonucleotide 5Ј-T TTA* GAG TCT GCT CCC-3Ј (Fig. 1D) has been reported (16). The other sequences shown in Fig.…”

“…The desired (10R) BPDE-adducted oligonucleotides were purified and separated from their (10S) diastereomers by reverse-phase HPLC (see Supporting Materials and Methods). Their absolute configurations were assigned by measurement of their CD spectra, which exhibit positive bands at 330 -350 nm in contrast to the (10S) diastereomers, which exhibit negative bands (16). In all cases, the (10R) diastereomers eluted before the (10S) diastereomers.…”

Efficiency and Accuracy of SOS-induced DNA Polymerases Replicating Benzo[a]pyrene-7,8-diol 9,10-Epoxide A and G Adducts

“…Replicative DNA polymerases are generally blocked by bulky adducts (26), and human pol ␣ replicates at best very poorly opposite BaP DE adducts (27), as does the non-SOS-induced E. coli polymerase I Klenow fragment (28,29). Thus, it is attractive to speculate that error-prone TLS by bypass polymerases may be highly significant in the observed induction of mutations by these adducts, especially in SOS-induced cells (30,31). Two central issues are addressed in this study.…”

Mutational Consequences on Replication of M13 Constructs Containing Isomeric Diol Epoxide Adducts in E. coli

“…The tabular portion of Figure 1 summarizes the total frequencies of substitution mutations observed on replication of the M13mp7L2 phage genome in SOS-induced E. coli (12)(13)(14)(15)(16). These adducts exhibit a wide variation in mutagenic response, ranging from essentially nil to as high as 68%-for the BaP DE-1 R cis dAdo adduct in context I(A).…”

“…Substitution mutations were scored by a differential hybridization assay as described (12). Depending on survival and mutational frequency, ∼90 to >14,000 plaques (more than 300 in most cases) were scored for each adduct (12)(13)(14)(15)(16). Results were verified by sequencing of selected mutants.…”