Lesion orientation of O4-alkylthymidine influences replication by human DNA polymerase η
DNA lesions that elude repair may undergo translesion synthesis catalyzed by Y-family DNA polymerases. O4-Alkylthymidines, persistent adducts that can result from carcinogenic agents, may be encountered by DNA polymerases. The influence of lesion orientation around the C4–O4 bond on processing by human DNA polymerase η (hPol η) was studied for oligonucleotides containing O4-methylthymidine (O4MedT), O4-ethylthymidine (O4EtdT), and analogs restricting the O4-methylene group in an anti-orientation. Primer extension assays revealed that the O4-alkyl orientation influences hPol η bypass. Crystal structures of hPol η·DNA·dNTP ternary complexes with O4MedT or O4EtdT in the template strand showed the nucleobase of the former lodged near the ceiling of the active site, with the syn-O4-methyl group engaged in extensive hydrophobic interactions. This unique arrangement for O4-methylthymidine with hPol η, inaccessible for the other analogs due to steric/conformational restriction, is consistent with differences observed for nucleotide incorporation and supports the concept that lesion conformation influences extension across DNA damage. Together, these results provide mechanistic insights on the mutagenicity of O4MedT and O4EtdT when acted upon by hPol η.