Endogenous metabolism, environmental exposure, and treatment with some cancer chemotherapeutic agents can give rise to DNA alkylation, which can occur on the phosphate backbone, forming alkylphosphotriester (PTE) lesions. Among the alkylated DNA lesions, PTEs were previously shown to display the longest half-life in cells, suggesting that they could be used as biomarkers for monitoring the long-term exposure to alkylating reagents and they are resistant to DNA repair. Recent research also showed that tobacco-specific nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), could be activated to form alkyl-PTE lesions in rat tissues. Our previous study revealed the influence of alkyl group size and stereochemistry of the phosphotriester on the replication and repair of alkyl-PTE lesions. It, however, remains elusive about the impact of flanking base sequences around PTE site on cellular DNA replication. In the present study, we synthesized oligodeoxyribonucleotides (ODNs) containing a site-specifically inserted methyl-PTE lesion, with different flanking base combinations (AT, CT, GT, TA, TC and TG). We also characterized the identities and purities of these ODNs by using LC-MS/MS analysis. Moreover, we assessed how these lesions inhibit DNA replication and induce mutations in Escherichia coli cells. We found that the three XT Sp-methylphosphotriesters were not strong impediments to DNA replication and replicative bypass of these lesions were mutagenic. Similar as what we observed previously for the Sp-T(Me)T phosphotriester, mutagenic bypass of these lesions requires Ada protein. On the other hand, the three Rp-methylphosphotriesters at XT site and all TX methylphosphotriesters moderately blocked DNA replication in E. coli, with no mutagenic products being detected.

Replicative bypass and mutagenic properties of alkylphosphotriester lesions in Escherichia coli

2019 Fall National Meeting

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