A wide range of endogenous and exogenous alkylating agents attack DNA to generate various alkylation adducts. N7-methyl-2-deoxyguanosine (Fm7dG) is the most abundant alkylative DNA lesion. If not repaired, Fm7dG can undergo spontaneous depurination, imidazole ring-opening, or bypass by translesion synthesis DNA polymerases. Human DNA polymerase η (polη) efficiently catalyzes across Fm7dG in vitro, but its structural basis is unknown. Herein, we report a crystal structure of polη in complex with templating Fm7dG and an incoming nonhydrolyzable dCTP analog, where a 2′-fluorine-mediated transition destabilization approach was used to prevent the spontaneous depurination of Fm7dG. The structure showed that polη readily accommodated the Fm7dG:dCTP base pair with little conformational change of protein and DNA. In the catalytic site, Fm7dG and dCTP formed three hydrogen bonds with a Watson–Crick geometry, indicating that the major keto tautomer of Fm7dG is involved in base pairing. The polη-Fm7dG:dCTP structure was essentially identical to the corresponding undamaged structure, which explained the efficient bypass of the major methylated lesion. Overall, the first structure of translesion synthesis DNA polymerase bypassing Fm7dG suggests that in the catalytic site of Y-family DNA polymerases, small N7-alkylguanine adducts may be well tolerated and form the canonical Watson–Crick base pair with dCTP through their keto tautomers.
Chronic inflammation is closely associated with cancer development. One possible mechanism for inflammation-induced carcinogenesis is DNA damage caused by reactive halogen species, such as hypochlorous acid, which is released by myeloperoxidase to kill pathogens. Hypochlorous acid can attack genomic DNA to produce 8-chloro-2′-deoxyguanosine (ClG) as a major lesion. It has been postulated that ClG promotes mutagenic replication using its syn conformer; yet, the structural basis for ClG-induced mutagenesis is unknown. We obtained crystal structures and kinetics data for nucleotide incorporation past a templating ClG using human DNA polymerase β (polβ) as a model enzyme for high-fidelity DNA polymerases. The structures showed that ClG formed base pairs with incoming dCTP and dGTP using its anti and syn conformers, respectively. Kinetic studies showed that polβ incorporated dGTP only 15-fold less efficiently than dCTP, suggesting that replication across ClG is promutagenic. Two hydrogen bonds between syn-ClG and anti-dGTP and a water-mediated hydrogen bond appeared to facilitate mutagenic replication opposite the major halogenated guanine lesion. These results suggest that ClG in DNA promotes G to C transversion mutations by forming Hoogsteen base pairing between syn-ClG and anti-G during DNA synthesis.
Non-enzymatic alkylation on DNA often generates N7-alkyl-2′-deoxyguanosine (N7alkylG) adducts as major lesions. N7alkylG adducts significantly block replicative DNA polymerases and can be bypassed by translesion synthesis (TLS) polymerases such as polymerase η (polη). To gain insights into the bypass of N7alkylG by TLS polymerases, we conducted kinetic and structural studies of polη catalyzing across N7BnG, a genotoxic lesion generated by the carcinogenic N-nitrosobenzylmethylamine. The presence of templating N7BnG in the polη catalytic site decreased the replication fidelity by ∼9-fold, highlighting the promutagenicity of N7BnG. The catalytic efficiency for dCTP incorporation opposite N7BnG decreased ∼22-fold and ∼7-fold compared to the incorporation opposite undamaged guanine in the presence of Mg2+ and Mn2+, respectively. A crystal structure of the complexes grown with polη, templating N7BnG, incoming dCTP, and Mg2+ ions showed the lack of the incoming nucleotide and metal cofactors in the polη catalytic site. Interestingly, the templating N7BnG adopted a syn conformation, which has not been observed in the published N7alkylG structures. The preferential formation of syn-N7BnG conformation at the templating site may deter the binding of an incoming dCTP, causing the inefficient bypass by polη. In contrast, the use of Mn2+ in place of Mg2+ in co-crystallization yielded a ternary complex displaying an anti-N7BnG:dCTP base pair and catalytic metal ions, which would be a close mimic of a catalytically competent state. We conclude that certain bulky N7-alkylG lesions can slow TLS polymerase-mediated bypass by adopting a catalytically unfavorable syn conformation in the replicating base pair site.
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