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.

中文翻译：

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的细胞毒性和在烷基磷酸三酯病变诱变性质大肠杆菌细胞

暴露于许多内源性和外源性物质会引起DNA烷基化，这是DNA损伤的主要类型。在DNA烷基化产物中，烷基磷酸三酯的出现频率较高，并且对哺乳动物组织的修复具有抵抗力。但是，关于这些损伤如何影响细胞中DNA复制的效率和保真度或如何通过跨病变合成DNA聚合酶调节这些损伤的复制旁路知之甚少。在这项研究中，我们合成了在定义的位点包含四对（S _p和R _p）烷基磷酸三酯病变的寡脱氧核糖核苷酸，并研究了这些病变如何被大肠杆菌中的DNA复制机制识别细胞。我们发现，烷基磷酸三酯病变的S _p非对映异构体可以被有效地绕过，而R _p对应物适度地阻断了DNA复制。而且，S _p-甲基磷酸三酯在侧翼的TT二核苷酸位点诱导TT→GT和TT→GC突变，并且这些突变的诱导需要Ada蛋白，已知该蛋白可有效地从S _p-甲基磷酸三酯中除去甲基。 。总之，我们的研究提供了对细胞中DNA复制机制对烷基磷酸三酯损伤识别的全面理解，并首次揭示了S端Ada依赖性突变的诱导。_对甲基磷酸三酯位点。