biostudies-literatureSanyal MRNational Institute of Environmental Health SciencesNIH National Institute of General Medical SciencesOhio UniversityNational Institutes of HealthInstitute of Biomedical Science110676https://www.ebi.ac.uk/biostudies/studies/S-EPMC12556801biostudies-literatureUnknown301(11)Temozolomide (TMZ), a DNA alkylator, is a chemotherapeutic agent for brain tumors, but the treatment induces a distinct pattern of mutations, known as a cancer mutational signature single-base substitution signature 11 (SBS11). Although the correlation between TMZ treatment and SBS11 mutations is very clear, the precise biochemical mechanisms that cause SBS11 have not been elucidated. TMZ can alkylate DNA at several locations, among which O⁶-methylguanine is believed to be the most toxic. In this study, we reconstituted potential biochemical processes of TMZ-induced mutagenesis in vitro, including TMZ-induced DNA damage and subsequent DNA synthesis by various DNA polymerases. Next-generation sequencing of the DNA products revealed mutations with a similar spectrum to SBS11. EffiThe Journal of biological chemistryBiochemical reconstitution of temozolomide-induced mutational processes.PMC12556801R15ES029723R15GM116098Sanyal MRSugiyama TfalseBiochemical reconstitution of temozolomide-induced mutational processes.Temozolomide (TMZ), a DNA alkylator, is a chemotherapeutic agent for brain tumors, but the treatment induces a distinct pattern of mutations, known as a cancer mutational signature single-base substitution signature 11 (SBS11). Although the correlation between TMZ treatment and SBS11 mutations is very clear, the precise biochemical mechanisms that cause SBS11 have not been elucidated. TMZ can alkylate DNA at several locations, among which O⁶-methylguanine is believed to be the most toxic. In this study, we reconstituted potential biochemical processes of TMZ-induced mutagenesis in vitro, including TMZ-induced DNA damage and subsequent DNA synthesis by various DNA polymerases. Next-generation sequencing of the DNA products revealed mutations with a similar spectrum to SBS11. Effi2025-01-01T00:00:00Z2025 Sep2026-06-05T05:53:06.893Z2026-05-13T14:29:16.315ZS-EPMC125568014091424510.1016/j.jbc.2025.110676