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

Authors

  • Alessia Stornetta,

    1. Department of Health Sciences and Technology, Institute of Food, Nutrition and Health, Swiss Federal Institute of Technology Zürich, Schmelzbergstrasse 9, 8092 Zürich (Switzerland)
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  • Dr. Todor Angelov,

    1. Department of Health Sciences and Technology, Institute of Food, Nutrition and Health, Swiss Federal Institute of Technology Zürich, Schmelzbergstrasse 9, 8092 Zürich (Switzerland)
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  • Prof. Dr. F. Peter Guengerich,

    1. Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, 2200 Pierce Avenue, Nashville, TN 37232-0146 (USA)
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  • Prof. Dr. Shana J. Sturla

    Corresponding author
    1. Department of Health Sciences and Technology, Institute of Food, Nutrition and Health, Swiss Federal Institute of Technology Zürich, Schmelzbergstrasse 9, 8092 Zürich (Switzerland)
    • Department of Health Sciences and Technology, Institute of Food, Nutrition and Health, Swiss Federal Institute of Technology Zürich, Schmelzbergstrasse 9, 8092 Zürich (Switzerland)===

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Abstract

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.

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