Alkyl phosphotriester (alkyl-PTE) lesions in DNA are shown to be poorly repaired; however, little is known about how these lesions impact DNA replication in human cells. Here, we investigated how the S_P and R_P diastereomers of four alkyl-PTE lesions (alkyl = Me, Et, nPr, or nBu) at the TT site perturb DNA replication in HEK293T cells. We found that these lesions moderately impede DNA replication and that their replicative bypass is accurate. Moreover, CRISPR-Cas9-mediated depletion of Pol η or Pol ζ resulted in significantly attenuated bypass efficiencies for both diastereomers of nPr- and nBu-PTE adducts, and the S_P diastereomer of Et-PTE. Diminished bypass efficiencies were also detected for the R_p diastereomer of nPr- and nBu-PTE lesions upon ablation of Pol κ. Together, our study uncovered the impact of the alkyl-PTE lesions on DNA replication in human cells and revealed the roles of individual translesion synthesis DNA polymerases in bypassing these lesions.

中文翻译：

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人体细胞中烷基磷酸三酯损伤的复制研究

DNA 中的烷基磷酸三酯（烷基-PTE）损伤修复不良；然而，人们对这些损伤如何影响人类细胞中 DNA 复制知之甚少。在这里，我们研究了TT 位点上四种烷基-PTE 损伤（烷基 = Me、Et、n Pr 或n _{Bu ）的}SP和R _{P非对映异构体如何扰乱 HEK293T 细胞中的 DNA 复制。}我们发现这些损伤会适度阻碍 DNA 复制，并且它们的复制旁路是准确的。此外，CRISPR-Cas9介导的Pol η或Pol ζ消耗导致n Pr-和n Bu-PTE加合物的非对映异构体以及Et-PTE的SP_非对映异构体的旁路效率显着减弱。消融 Pol κ 后，还检测到n Pr- 和n Bu-PTE 病变的R _p非对映异构体的旁路效率降低。总之，我们的研究揭示了烷基 PTE 损伤对人类细胞 DNA 复制的影响，并揭示了个体跨损伤合成 DNA 聚合酶在绕过这些损伤中的作用。