Sequence Specific Mutagenesis of the Major (+)-anti-Benzo[a]pyrene Diol Epoxide−DNA Adduct at a Mutational Hot Spot in Vitro and in Escherichia coli Cells

Abstract

In the supF gene, most (+)-anti-benzo[a]pyrene diol epoxide ((+)-anti-B[a]PDE) mutagenesis hot spots in Escherichia coli are in 5'-GG sequences [Rodriguez and Loechler (1993) Carcinogenesis 14, 373-383]. A major hot spot was detected at G1 in the sequence 5'-GCG1G2-CCAAAG, whereas G2 yielded very few mutants. In order to investigate the details of such sequence context effects of (+)-anti-B[a]PDE mutagenesis, we have constructed 25-mer oligonucleotides and single-stranded M13 genomes containing the above decamer sequence, in which the trans-N2-dG adduct induced by (+)-anti-B[a]PDE [(+)-trans-anti-B[a]P-N2-dG] at G1 or G2 was introduced. In vitro DNA synthesis on the adducted 25-mers was strongly blocked at each site, although the 3'-->5' exonuclease-deficient Klenow fragment could incorporate a nucleotide opposite the adduct in the presence of Mn2+. For both sites purine nucleotides were preferred. The ratio Vmax/K(m) indicated that the efficiency of incorporation of dGTP opposite these sites was very similar, but dATP incorporation opposite the adduct at G1 was five-fold more efficient than that at G2. For each site, further extension beyond the adducted nucleotide was investigated by annealing four different primers, in which only the nucleotide opposite the adducted deoxyguanosine was altered. Significant extension was only observed when deoxyadenosine was located opposite adducted G1. When the M13 genomes containing the (+)-trans-anti-B[a]P-N2-dG were replicated in E. coli, survival of each adducted genome was less than 1% as compared to the unadducted genome. Upon induction of SOS, viability increased 2-6-fold. DNA sequencing showed no base substitutions in the progeny from SOS-uninduced cells, although small deletions in a quasipalindromic sequence occurred with the adduct being located at either site. However, following SOS induction, up to 40% targeted base substitutions were detected when the adduct was located at G1, while approximately 12% of the progeny were mutants with the adduct at G2. Most base substitutions were targeted G-->T transversions. We conclude that (+)-trans-anti-B[a]P-N2-dG is a highly mutagenic and replication blocking lesion. In addition, the biological consequence of this adduct depends on whether it is located at G1 or G2, suggesting that sequence context plays a major role in the mutagenic processing of this adduct.

Light‐dependent mutagenesis by benzo[a]pyrene is mediated via oxidative DNA damage

“…Although the repair mechanism for dIz in DNA is poorly understood, it seems reasonable to assume that deletion of mutM ST increases the amount of dIz in DNA, at least in part, by increasing the level of 8-oxoG in DNA. The mutational spectra ( sequences, with more mutations occurring at the 5 0 -G than at the 3 0 -G [Rodriguez and Loechler, 1993;Hanrahan et al, 1997]. This is consistent with our findings that G 2 is more susceptible to metabolically activated B[a]P than G 3 .…”

High-fidelity in vivo replication of DNA base shape mimics without Watson–Crick hydrogen bonds

“…The ability of the F and Q isosteres to block DNA replication in vivo was addressed by using a common phage-survival assay (19)(20)(21)(22)(23). Briefly, genomes were constructed either bearing or lacking a probe isostere (lesion), and an equal amount of each genome was transfected into E. coli.…”

Quantitative Analysis of Translesion DNA Synthesis across a Benzo[a]pyrene-Guanine Adduct in Mammalian Cells

“…Similar results were obtained with plasmid GP-BPG2 (kan R ) (52 Ϯ 4%; Table I (top)). This extent of bypass is much higher than values usually reported for bypass across BP-G adducts in E. coli (37)(38)(39). However, since the differences might stem from DNA sequence context effects, which are known to strongly affect bypass across BP-G adducts in vitro and in E. coli in vivo (40 -43), we assayed TLS across the BP-G adduct in E. coli cells, using the same gapped plasmids used in the mammalian cells.…”