Sequence Context Profoundly Influences the Mutagenic Potency of Trans-Opened Benzo[a]pyrene 7,8-Diol 9,10-Epoxide−Purine Nucleoside Adducts in Site-Specific Mutation Studies

Abstract

The postoligomerization method was used to prepare oligonucleotide 16-mers that contained dAdo or dGuo adducts, derived from trans opening of each enantiomer of the two diastereomeric benzo[a]pyrene 7,8-diol 9,10-epoxides, in two sequence contexts. These 16 oligonucleotides, along with the four corresponding oligonucleotides containing unsubstituted purines, were ligated into single-stranded DNA from bacteriophage M13mp7L2 and transfected into Escherichia coli SMH77. The mutagenic effects of replication past these adducts were then evaluated. The various adduct isomers induced point mutations at different frequencies and with different distributions of mutation types, as was anticipated. However, sequence context had the most substantial effects on mutation frequency. A high frequency of deletions of a single guanine was found in a context where the dGuo adduct was at the 3'-end of a run of five guanines, whereas no single base deletion was found in the other context studied, 5'-CGA-3'. Mutation frequencies in constructs containing dAdo adducts were much higher in a 5'-TAG-3' context (37-58%, depending on the individual isomer) than in a 5'-GAT-3' context (5-20%), and for a given adduct, mutation frequency was up to 10-fold higher in the former sequence than in the latter. These findings indicate that sequence context effects need more thorough evaluation if the goal of understanding the mechanism through which DNA adducts lead to mutation is to be achieved.

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

“…The guanine base may have to shift toward the minor groove (33), thus inducing mispairing with dT or dA. In support of this hypothesis, it has been reported that, depending on the sequence context, BPDE-dG adducts induce either G 3 A or G 3 T mutations (30,31).…”

“…The unusually small finger and thumb domains of the Y-family polymerases leave the major and minor groove wide open (26)(27)(28)(29) and thus may facilitate such adduct placement. The crystal structure, although representing primer extension beyond the dA* lesion, offers insights into how base substitution and frameshift mutations are induced by BPDE adducts at dA and dG (30)(31)(32). When the PAH is placed in the major groove in the BP-2 complex, the adenine base of the dA* shifts Ϸ2 Å toward the major groove, juxtaposing two hydrogen bond acceptors, the N1 of dA* and the O 4 of its base pair partner dT (Fig.…”

Position-specific Suppression and Enhancement of HIV-1 Integrase Reactions by Minor Groove Benzo[a]pyrene Diol Epoxide Deoxyguanine Adducts

“…1A) were prepared from the mixed 10R/ 10S diastereomers of the BaP DE dG phosphoramidite (20) and were separated by reverse-phase high-performance liquid chromatography. Their absolute configurations were assigned from the long-wavelength CD spectra of the separated oligonucleotides, which exhibit positive bands in the 330 -360 nm region for the 10R adduct and negative bands for the 10S adduct (21). All other adducted oligonucleotides were prepared from diastereomerically pure 10R or 10S phosphoramidites derived from the 10R and 10S diastereomers of the BaP DE dG adducts that had been separated as their O 6 -allyl di-tert-butyldimethylsilyl triacetates (22).…”

Preferential Misincorporation of Purine Nucleotides by Human DNA Polymerase η Opposite Benzo[a]pyrene 7,8-Diol 9,10-Epoxide Deoxyguanosine Adducts

“…Oligonucleotides-Oligonucleotides containing a single specific cis or trans BaP DE-2 dG adduct placed on the fourth nucleotide from the 5Ј-end in either sequence context III(G) (5Ј-TTCG*AATCCTTCCCCC-3Ј) or IV(G) (5Ј-GGGG*TTCCCGAGCGGC-3Ј) were synthesized as described previously (29,30). The basis for selection of these sequences has been described previously (30).…”