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N(6)-(2-Deoxy-d-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine (MeFapy-dGuo) has been identified as a stable DNA adduct that arises from the reaction of DNA with a variety of methylating agents. Since this lesion persists in DNA and may contribute to the overall mutagenesis from electrophilic methylating agents, the MeFapy-dGuo lesion was incorporated into oligonucleotides, and its replication bypass was examined in vitro with a panel of eukaryotic high fidelity (hPols α, β, and δ/PCNA) and translesion (hPols η, κ, ι, Rev1, ν, and yPol ζ) polymerases to address its miscoding potential. The MeFapy-dGuo was found to be a strong block to the high fidelity polymerases at either the insertion or the extension step. Efficient translesion synthesis was observed for hPols η and κ, and the combined activities of hRev1 and yPol ζ. The nucleotide sequences of the extension products were determined by mass spectrometry. The error-free extension product was the most abundant product observed for each polymerase. Misreplication products, which included misinsertion of Thy, Gua, and Ade opposite the MeFapy-dGuo lesion, as well as an interesting one-nucleotide deletion product, were observed when hPols η and κ were employed; these events accounted for 8-29% of the total extension products observed. The distribution and abundance of the misreplication products were dependent on the polymerases and local sequence context of the lesion. Collectively, these data suggest that although MeFapy-dGuo adducts represent a relatively minor proportion of the total alkylated lesions, their miscoding potentials could significantly contribute to genomic instability.