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, 14 (13), 1634-9

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

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

Alessia Stornetta et al. Chembiochem.

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.

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

Figures

Figure 1
Figure 1
Single primer extension with unmodified and O6-MeG-containing template catalysed by Dpo4 at 37 °C with magnesium in the reaction buffer. Oligonucleotide product length is shown on the right of the gel image. The reaction time was 45 min. Final [dNTP]G: 400 µM, final [dNTP]O6-MeG: 600 or 800 µM.
Figure 2
Figure 2
Selectivity factor of steady-state kinetic parameters for dCTP, 5-Me-dCTP, 2-thio-dCTP, or dTTP for incorporation opposite O6-MeG versus G. The selectivity factor is calculated as (kcat/Km)O6-MeG/(kcat/Km)G. A selectivity factor > 1 means that the dNTP is preferentially incorporated opposite O6-MeG. aCalculated as the ratio between (kcat/Km)O6-MeG and (kcat/Km)G reported by Eoff et al.[9]
Scheme 1
Scheme 1
Wobble O6-MeG:C (left) and pseudo-Watson-Crick O6-MeG:T base pairs (right). R=deoxyribose in DNA strand.[9]
Scheme 2
Scheme 2
Natural and non-natural dNTPs evaluated in this study. dR= deoxyribose triphosphate.

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