Differential mutagenic patterns were recently reported for O-methylated thymine lesions, which indicate that O4methylthymine (O4-Me-T) frequently leads to G misinsertions, whereas O2-methylthymine (O2-Me-T) is primarily nonmutagenic. The reasons for these differences are unclear since both lesions similarly alter the Watson−Crick binding face of T. To rationalize these replication outcomes at a molecular level, this work uses density functional theory calculations and molecular dynamics simulations to probe the lesion base-pairing properties as well as lesion accommodation by human polymerase η (pol η) and postextension DNA duplexes. O4-Me-T forms two strong hydrogen bonds with an opposing G in the active site of pol η, which rationalizes the observed lesion mutagenicity. Nevertheless, dATP insertion opposite O4-Me-T can proceed through water-mediated hydrogen bonding, which is similar to the pathway previously proposed for pol η bypass of abasic sites and other T alkylation lesions. In contrast, the position of O2-Me-T in the pol η active site is dynamic due to the presence of the aberrant methyl group on the minor groove side of DNA. In fact, the experimental replication outcomes can only be rationalized when the syn glycosidic orientation of O2-Me-T is considered, which stabilizes the pre-insertion complex by placing the damage in the polymerase open pocket on the major groove side of DNA. Although dATP insertion can occur opposite syn-O2-Me-T through a water-mediated pathway similar to O4-Me-T replication, rotation about the glycosidic bond precludes a stable pol η ternary complex corresponding to dGTP insertion, which correlates with the reported nonmutagenic bypass of O2-Me-T. In addition to providing structural insights into the differential mutagenicity of methylated T adducts, our data highlight an emerging theme in the literature for the replication of pyrimidine alkylation products in noncanonical glycosidic orientations and sets the stage for future work on the replication of other alkylated lesions by TLS polymerases.

Tobacco-derived pyridyloxobutyl (POB) DNA adducts are unique due to the large size and flexibility of the alkyl chain connecting the pyridyl ring to the nucleobase. Recent experimental work suggests that the O4-4-(3-pyridyl)-4-oxobut-1-yl-T (O4-POB-T) lesion can undergo both nonmutagenic (dATP) and mutagenic (dGTP) insertion by the translesion synthesis (TLS) polymerase (pol) η in human cells. Interestingly, the mutagenic rate for O4-POB-T replication is reduced compared to that for the smaller O4-methylthymine (O4-Me-T) lesion, and O4-POB-T yields a different mutagenic profile than the O2-POB-T variant (dTTP insertion). The present work uses a combination of density functional theory calculations and molecular dynamics simulations to probe the impact of the size and flexibility of O4-POB-T on pol η replication outcomes. Due to changes in the Watson–Crick binding face upon damage of canonical T, O4-POB-T does not form favorable hydrogen-bonding interactions with A. Nevertheless, dATP is positioned for insertion in the pol η active site by a water chain to the template strand, which suggests a pol η replication pathway similar to that for abasic sites. Although a favorable O4-POB-T:G mispair forms in the pol η active site and DNA duplexes, the inherent dynamical nature of O4-POB-T periodically disrupts interstrand hydrogen bonding that would otherwise facilitate dGTP insertion and stabilize damaged DNA duplexes. In addition to explaining the origin of the experimentally reported pol η outcomes associated with O4-POB-T replication, comparison to structural data for the O4-Me-T and O2-POB-T adducts highlights an emerging common pathway for the nonmutagenic replication of thymine alkylated lesions by pol η, yet underscores the broader impacts of bulky moiety size, flexibility, and position on the associated mutagenic outcomes.

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