Cytotoxic and mutagenic properties of alkyl phosphotriester lesions in Escherichia coli cells

Abstract

Exposure to many endogenous and exogenous agents can give rise to DNA alkylation, which constitutes a major type of DNA damage. Among the DNA alkylation products, alkyl phosphotriesters have relatively high frequencies of occurrence and are resistant to repair in mammalian tissues. However, little is known about how these lesions affect the efficiency and fidelity of DNA replication in cells or how the replicative bypass of these lesions is modulated by translesion synthesis DNA polymerases. In this study, we synthesized oligodeoxyribonucleotides containing four pairs ( S _p and R _p) of alkyl phosphotriester lesions at a defined site, and examined how these lesions are recognized by DNA replication machinery in Escherichia coli cells. We found that the S _p diastereomer of the alkyl phosphotriester lesions could be efficiently bypassed, whereas the R _p counterparts moderately blocked DNA replication. Moreover, the S _p-methyl phosphotriester induced TT→GT and TT→GC mutations at the flanking TT dinucleotide site, and the induction of these mutations required Ada protein, which is known to remove efficiently the methyl group from the S _p-methyl phosphotriester. Together, our study provided a comprehensive understanding about the recognition of alkyl phosphotriester lesions by DNA replication machinery in cells, and revealed for the first time the Ada-dependent induction of mutations at the S _p-methyl phosphotriester site.