Roles of Aag, Alkbh2, and Alkbh3 in the Repair of Carboxymethylated and Ethylated Thymidine Lesions

ACS Chem Biol. 2016 May 20;11(5):1332-8. doi: 10.1021/acschembio.6b00085. Epub 2016 Mar 4.

Abstract

Environmental and endogenous genotoxic agents can result in a variety of alkylated and carboxymethylated DNA lesions, including N3-ethylthymidine (N3-EtdT), O(2)-EtdT, and O(4)-EtdT as well as N3-carboxymethylthymidine (N3-CMdT) and O(4)-CMdT. By using nonreplicative double-stranded vectors harboring a site-specifically incorporated DNA lesion, we assessed the potential roles of alkyladenine DNA glycosylase (Aag); alkylation repair protein B homologue 2 (Alkbh2); or Alkbh3 in modulating the effects of N3-EtdT, O(2)-EtdT, O(4)-EtdT, N3-CMdT, or O(4)-CMdT on DNA transcription in mammalian cells. We found that the depletion of Aag did not significantly change the transcriptional inhibitory or mutagenic properties of all five examined lesions, suggesting a negligible role of Aag in the repair of these DNA adducts in mammalian cells. In addition, our results revealed that N3-EtdT, but not other lesions, could be repaired by Alkbh2 and Alkbh3 in mammalian cells. Furthermore, we demonstrated the direct reversal of N3-EtdT by purified human Alkbh2 protein in vitro. These findings provided important new insights into the repair of the carboxymethylated and alkylated thymidine lesions in mammalian cells.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase / metabolism*
  • AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase / metabolism*
  • Alkylation
  • Animals
  • Cell Line
  • DNA Adducts / chemistry
  • DNA Adducts / genetics
  • DNA Adducts / metabolism*
  • DNA Glycosylases / metabolism*
  • DNA Repair
  • Humans
  • Mice
  • Thymidine / analogs & derivatives
  • Thymidine / chemistry
  • Thymidine / genetics
  • Thymidine / metabolism

Substances

  • DNA Adducts
  • N3-carboxymethylthymidine
  • N(3)-ethylthymidine
  • ALKBH3 protein, human
  • AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase
  • Alkbh3 protein, mouse
  • ALKBH2 protein, human
  • AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase
  • Alkbh2 protein, mouse
  • 3-methyladenine-DNA glycosylase
  • DNA Glycosylases
  • Thymidine