TY - JOUR
T1 - Incorporation of Nucleoside Probes Opposite O6-Methylguanine by Sulfolobus solfataricus DNA Polymerase Dpo4
T2 - Importance of Hydrogen Bonding
AU - Stornetta, Alessia
AU - Angelov, Todor
AU - Guengerich, F. Peter
AU - Sturla, Shana J.
PY - 2013/9
Y1 - 2013/9
N2 - 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.
AB - 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.
KW - DNA damage
KW - DNA polymerases
KW - Fluorescence
KW - Nucleotide analogues
KW - Translesion DNA synthesis
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U2 - 10.1002/cbic.201300296
DO - 10.1002/cbic.201300296
M3 - Article
C2 - 23959784
AN - SCOPUS:84883196741
SN - 1439-4227
VL - 14
SP - 1634
EP - 1639
JO - ChemBioChem
JF - ChemBioChem
IS - 13
ER -