title: Incorporation of nucleoside probes opposite O⁶-methylguanine by Sulfolobus solfataricus DNA polymerase Dpo4: importance of hydrogen bonding.
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.
authors: Department of Health Sciences and Technology, Institute of Food, Nutrition and Health, Swiss Federal Institute of Technology Zürich, Schmelzbergstrasse 9, 8092 Zürich (Switzerland).
authors: Alessia
authors: A
authors: Stornetta
authors: Todor
authors: T
authors: Angelov
authors: F Peter
authors: FP
authors: Guengerich
authors: Shana J
authors: SJ
authors: Sturla
mesh_terms: D019951:DNA Polymerase beta / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006860:Hydrogen Bonding; D015394:Molecular Structure; D009705:Nucleosides / Q000737:chemistry* / Q000378:metabolism; D048229:Sulfolobus solfataricus / Q000201:enzymology*
chemical_list: D005456:Fluorescent Dyes; D009705:Nucleosides; D006147:Guanine; C008449:O-(6)-methylguanine; D019951:DNA Polymerase beta
keywords: DNA damage; DNA polymerases; fluorescence; nucleotide analogues; translesion DNA synthesis
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