Incorporation of Nucleoside Probes Opposite O6-Methylguanine by Sulfolobus solfataricus DNA Polymerase Dpo4: Importance of Hydrogen Bonding

Alessia Stornetta, Todor Angelov, F. Peter Guengerich, Shana J. Sturla

Research output: Contribution to journalArticlepeer-review

8 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)1634-1639
Number of pages6
Issue number13
StatePublished - Sep 2013
Externally publishedYes


  • DNA damage
  • DNA polymerases
  • Fluorescence
  • Nucleotide analogues
  • Translesion DNA synthesis


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