The effects of N7-methylguanine on duplex DNA structure.

Non-enzymatic methylation of DNA by endogenous and exogenous agents produces a variety of adducts, of which the predominant one is N7-methyl-2'-deoxyguanosine (m7dG). Although it is known that living organisms counter the deleterious effects of m7dG by producing adduct-specific DNA repair proteins, the molecular basis for specific recognition and catalysis by these proteins is poorly understood. In addition to its role as an endogenous DNA adduct, m7dG is also widely used as an in vitro probe of protein-DNA interactions. We set out to examine whether incorporation of m7dG into DNA affects duplex DNA structure. We carried out a large-scale synthesis of a dodecamer containing the m7dG adduct at a single, defined position. Because the instability of m7dG precludes its incorporation into oligonucleotides by standard solid-phase methods, a novel strategy employing chemical and enzymatic synthesis was used. Characterization of the m7dG-containing dodecamer by NMR reveals no structural distortion; indeed, m7dG appears to encourage a modest shift toward a more characteristically B-form duplex. These results argue strongly against induced DNA distortion as a mechanism for specific recognition of m7dG by adduct-specific repair proteins. The broad substrate specificity of these repair proteins disfavors a model involving direct recognition of aberrantly placed methyl groups; hence, it may be that m7dG is recognized indirectly, perhaps by its effects on the dynamics of DNA. On the other hand, the evidence presented here suggests that m7dG interferes directly with sequence-specific recognition by DNA-binding proteins by steric blockage or by masking of required contact functionalities. The synthetic methodology used here should be generally applicable to high-resolution structural studies of oligonucleotides bearing adducts that are unstable to the conditions of solid-phase DNA synthesis.