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Published on: March 5, 2018
Replication Studies of Alkyl Phosphotriester Lesions in Human Cells
Jun Wu1, Jiabin Wu2, Garrit Clabaugh1
1Department of Chemistry, University of California Riverside, Riverside, California 92521-0403, United States.
Abstract:
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 SP and RP 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 SP diastereomer of Et-PTE. Diminished bypass efficiencies were also detected for the Rp 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.
Insights
Alkyl phosphotriester (alkyl-PTE) DNA lesions moderately slow replication but are bypassed accurately. Specific translesion synthesis polymerases (Pol η, Pol ζ, Pol κ) are crucial for bypassing these DNA damage types.
Area of Science:
- Molecular Biology
- DNA Repair
- Genetics
Background:
- Alkyl phosphotriester (alkyl-PTE) lesions are DNA damage types that are poorly repaired.
- The impact of alkyl-PTE lesions on DNA replication in human cells remains largely unknown.
Purpose of the Study:
- To investigate how alkyl-PTE lesions affect DNA replication in human cells.
- To identify the roles of translesion synthesis (TLS) polymerases in bypassing alkyl-PTE lesions.
Main Methods:
- Investigated the impact of four alkyl-PTE lesions (Me, Et, nPr, nBu) and their diastereomers on DNA replication in HEK293T cells.
- Utilized CRISPR-Cas9 to deplete TLS polymerases (Pol η, Pol ζ, Pol κ) and assessed bypass efficiencies.
Main Results:
- Alkyl-PTE lesions moderately impede DNA replication, with accurate bypass observed.
- Depletion of Pol η or Pol ζ significantly reduced bypass of nPr- and nBu-PTE adducts, and SP-Et-PTE.
- Ablation of Pol κ diminished bypass of Rp-nPr- and Rp-nBu-PTE lesions.
Conclusions:
- This study elucidates the effects of alkyl-PTE lesions on DNA replication in human cells.
- Identified specific TLS polymerases (Pol η, Pol ζ, Pol κ) involved in the accurate bypass of various alkyl-PTE DNA damage types.
