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Replication studies of carboxymethylated DNA lesions in human cells

Jun Wu1, Pengcheng Wang2, Lin Li1

  • 1Department of Chemistry, University of California, Riverside, CA 92521, USA.

Nucleic Acids Research
|May 23, 2017
PubMed

Insights

Carboxymethylated DNA lesions like O6-CMdG, N3-CMdT, and O4-CMdT block DNA replication and cause mutations. Specific DNA polymerases (Pol η, κ, ζ) help bypass these lesions, impacting cancer mutations.

Area of Science:

  • Molecular Biology
  • Genetics
  • Toxicology

Background:

  • N-nitroso compounds (NOCs) are DNA damaging agents.
  • Metabolic activation of NOCs leads to DNA carboxymethylation.
  • The impact of carboxymethylated DNA lesions on human DNA replication is poorly understood.

Purpose of the Study:

  • Investigate the effects of five carboxymethylated DNA lesions on DNA replication efficiency and fidelity in human cells.
  • Determine the roles of translesion synthesis (TLS) DNA polymerases in bypassing these lesions.
  • Correlate specific lesions with mutations in the p53 gene in human cancers.

Main Methods:

  • Utilized HEK293T human embryonic kidney cells.
  • Assessed DNA replication efficiency and fidelity following exposure to O6-CMdG, N6-CMdA, N4-CMdC, N3-CMdT, and O4-CMdT.
  • Employed CRISPR-Cas9 to deplete specific TLS DNA polymerases (Pol η, Pol κ, Pol ζ).

Main Results:

  • N6-CMdA and N4-CMdC did not block replication or induce mutations.
  • N3-CMdT, O4-CMdT, and O6-CMdG moderately blocked replication and induced significant T→A, T→C, and G→A mutations, respectively.
  • Depletion of Pol η reduced bypass of N4-CMdC and N3-CMdT; Pol κ and Pol ζ depletion affected N6-CMdA, O6-CMdG bypass.

Conclusions:

  • Carboxymethylated DNA lesions differentially impact DNA replication and fidelity.
  • TLS polymerases play distinct roles in bypassing these lesions.
  • O6-CMdG, N3-CMdT, and O4-CMdT are implicated in p53 mutations in gastrointestinal cancers.

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