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Safrole is a natural product present in many plants and plant products, including spices and essential oils. During cellular metabolism, it converts to a highly reactive trans-isosafrole (SF) intermediate that reacts with genomic DNA and forms N 2 -SF-dA DNA adducts, which are detected in the oral tissue of cancer patients with betel quid chewing history. To study the SF-induced carcinogenesis and to probe the role of low fidelity translesion synthesis (TLS) polymerases in bypassing SF adducts, herein, we report the synthesis of N 6 -SF-dA DNA adducts, which are detected in the oral tissue of cancer patients with betel quid chewing history. To study the SF-induced carcinogenesis and to probe the role of low fidelity translesion synthesis (TLS) polymerases in bypassing SF adducts, herein, we report the synthesis of N 2 -SF-dG modified DNAs using phosphoramidite chemistry. The N 2 -SF-dG modification in the duplex DNA does not affect the thermal stability and retains the B-form of helical conformation, indicating that this adduct may escape the radar of common DNA repair mechanisms. Primer extension studies showed that the N 2 -SF-dG adduct is bypassed by human TLS polymerases hpolκ and hpolη, which perform error-free replication across this adduct. Furthermore, molecular modeling and dynamics studies revealed that the adduct reorients to pair with the incoming nucleotide, thus allowing the effective bypass. Overall, the results indicate that hpolκ and hpolη do not distinguish the N 2 -SF-dG adduct, suggesting that they may not be involved in the safrole-induced carcinogenicity.

The secondary metabolites of polypropanoids, methyleugenol (MEG), and estragole (EG), found in many herbs and spices, are commonly used as food flavoring agents and as ingredients in cosmetics. MEG and EG have been reported to cause hepatocarcinogenicity in rodents, human livers, and lung cells. The formation of N 2 -dA DNA adducts is primarily attributed to the carcinogenicity of these compounds. Therefore, these compounds have been classified as "possible human carcinogens" by the International Agency for Research on Cancer and "reasonably anticipated to be a human carcinogen" by the National Toxicology Program. Herein, we report the synthesis of the N 6 -EG-dG modified oligonucleotides to study the mutagenicity of these DNA adducts. Our studies show that N 2 -MEG-dG and N 2 -EG-dG modified oligonucleotides to study the mutagenicity of these DNA adducts. Our studies show that N 2 -MEG-dG and N 2 -EG-dG could be bypassed by human translesion synthesis (TLS) polymerases hpolκ and hpolη in an error-free manner. The steady-state kinetics of dCTP incorporation by hpolκ across N 2 -MEG-dG and N 2 -EG-dG adducts show that the catalytic efficiencies ( k cat / K m ) were ∼2.5- and ∼4.4-fold higher, respectively, compared to the unmodified dG template. A full-length primer extension assay demonstrates that hpolκ exhibits better catalytic efficiency than hpolη. Molecular modeling and dynamics studies capturing pre-insertion, insertion, and post-insertion steps reveal the structural features associated with the efficient bypass of the N 2 -MEG-dG adduct by hpolκ and indicate the reorientation of the adduct in the active site allowing the successful insertion of the incoming nucleotide. Together, these results suggest that though hpolκ and hpolη perform error-free TLS across MEG and EG during DNA replication, the observed carcinogenicity of these adducts could be attributed to the involvement of other low fidelity polymerases.