Journal
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
Volume 144, Issue 18, Pages 8054-8065Publisher
AMER CHEMICAL SOC
DOI: 10.1021/jacs.2c00193
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- National Institute of Environmental Health Sciences [ES-027558]
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This study presents the crystallographic characterization of Fapy•dG in duplex DNA and as the template base for DNA polymerase beta (Pol β). Fapy•dG adopts different anomers depending on the base pairing, and ring opening and epimerization occur in the crystalline state. The mutagenicity of Fapy•dG is influenced by Pol β, resulting in a decrease in polymerase fidelity. The kinetic studies reveal that dCMP incorporation opposite the beta-anomer is significantly faster than the alpha-anomer.
N6-(2-Deoxy-alpha,beta-D-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamido pyrimidine (Fapy center dot dG) is a prevalent form of genomic DNA damage. Fapy center dot dG is formed in greater amounts under anoxic conditions than the well-studied, chemically related 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodGuo). Fapy center dot dG is more mutagenic in mammalian cells than 8-oxodGuo. A distinctive property of Fapy center dot dG is facile epimerization, but prior works with Fapy center dot dG analogues have precluded determining its effect on chemistry. We present crystallographic characterization of natural Fapy center dot dG in duplex DNA and as the template base for DNA polymerase beta (Pol beta). Fapy center dot dG adopts the beta-anomer when base paired with cytosine but exists as a mixture of alpha- and beta-anomers when promutagenically base paired with adenine. Rotation about the bond between the glycosidic nitrogen atom and the pyrimidine ring is also affected by the opposing nucleotide. Sodium cyanoborohydride soaking experiments trap the ring-opened Fapy center dot dG, demonstrating that ring opening and epimerization occur in the crystalline state. Ring opening and epimerization are facilitated by propitious water molecules that are observed in the structures. Determination of Fapy center dot dG mutagenicity in wild type and Pol beta knockdown HEK 293T cells indicates that Pol beta contributes to G -> T transversions but also suppresses G -> A transitions. Complementary kinetic studies have determined that Fapy center dot dG promotes mutagenesis by decreasing the catalytic efficiency of dCMP insertion opposite Fapy center dot dG, thus reducing polymerase fidelity. Kinetic studies have determined that dCMP incorporation opposite the beta-anomer is similar to 90 times faster than the alpha-anomer. This research identifies the importance of anomer dynamics, a feature unique to formamidopyrimidines, when considering the incorporation of nucleotides opposite Fapy center dot dG and potentially the repair of this structurally unusual lesion.
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