Replication Studies of Alkyl Phosphotriester Lesions in Human Cells.

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, n Pr, or n Bu) 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 n Pr- and n Bu-PTE adducts, and the S P diastereomer of Et-PTE. Diminished bypass efficiencies were also detected for the R p diastereomer of n Pr- and n Bu-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.