Synthesis and Structure of Duplex DNA Containing the Genotoxic Nucleobase Lesion N7-Methylguanine

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Abstract
The predominant product of aberrant DNA methylation is the genotoxic lesion N7-methyl-2′-deoxyguanosine (m7dG). M7dG is recognized and excised by lesion-specific DNA glycosylases, namely AlkA in E. coli and Aag in humans. Structural studies of m7dG recognition and catalysis by these enzymes have been hampered due to a lack of efficient means by which to incorporate the chemically labile m7dG moiety site-specifically into DNA on a preparative scale. Here we report a solution to this problem. We stabilized the lesion toward acid-catalyzed and glycosylase-catalyzed depurination by 2′-fluorination and toward base-catalyzed degradation using mild, nonaqueous conditions in the DNA deprotection reaction. Duplex DNA containing 2′-fluoro-m7dG (Fm7dG) cocrystallized with AlkA as a host−guest complex in which the lesion-containing segment of DNA was nearly devoid of protein contacts, thus enabling the first direct visualization of the N7-methylguanine lesion nucleobase in DNA. The structure reveals that the base-pairing mode of Fm7dG:C is nearly identical to that of G:C, and Fm7dG does not induce any apparent structural disturbance of the duplex structure. These observations suggest that AlkA and Aag must perform a structurally invasive interrogation of DNA in order to detect the presence of intrahelical m7dG lesions.