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, O⁴-ethylthymidine, 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 O⁴-methyl- or O⁴-ethylthymidine 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.