Incorporation of nucleoside probes opposite O⁶-methylguanine by Sulfolobus solfataricus DNA polymerase Dpo4: importance of hydrogen bonding

Chembiochem. 2013 Sep 2;14(13):1634-9. doi: 10.1002/cbic.201300296. Epub 2013 Aug 19.


O⁶-Methylguanine (O⁶-MeG) is a mutagenic DNA lesion, arising from the action of methylating agents on guanine (G) in DNA. Dpo4, an archaeal low-fidelity Y-family DNA polymerase involved in translesion DNA synthesis (TLS), is a model for studying how human Y-family polymerases bypass DNA adducts. Previous work showed that Dpo4-mediated dTTP incorporation is favored opposite O⁶-MeG rather than opposite G. However, factors influencing the preference of Dpo4 to incorporate dTTP opposite O⁶-MeG are not fully defined. In this study, we investigated the influence of structural features of incoming dNTPs on their enzymatic incorporation opposite O⁶-MeG in a DNA template. To this end, we utilized a new fluorescence-based primer extension assay to evaluate the incorporation efficiency of a panel of synthetic dNTPs opposite G or O⁶-MeG by Dpo4. In single-dNTP primer extension studies, the synthetic dNTPs were preferentially incorporated opposite G, relative to O⁶-MeG. Moreover, pyrimidine-based dNTPs were generally better incorporated than purine-based syn-conformation dNTPs. The results suggest that hydrophobicity of the incoming dNTP appears to have little influence on the process of nucleotide selection by Dpo4, with hydrogen bonding capacity being a major influence. Additionally, modifications at the C2-position of dCTP increase the selectivity for incorporation opposite O⁶-MeG without a significant loss of efficiency.

Keywords: DNA damage; DNA polymerases; fluorescence; nucleotide analogues; translesion DNA synthesis.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Polymerase beta / metabolism*
  • Fluorescent Dyes / chemistry*
  • Fluorescent Dyes / metabolism
  • Guanine / analogs & derivatives*
  • Guanine / chemistry
  • Guanine / metabolism
  • Hydrogen Bonding
  • Molecular Structure
  • Nucleosides / chemistry*
  • Nucleosides / metabolism
  • Sulfolobus solfataricus / enzymology*


  • Fluorescent Dyes
  • Nucleosides
  • Guanine
  • O-(6)-methylguanine
  • DNA Polymerase beta