Biochemical 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. Efficient production of the SBS11-like mutation spectra required DNA in double-stranded form and multiple exposures to TMZ. Replicative polymerase δ alone generated SBS11-like mutations on the damaged DNA. Most SBS11-like mutations were sensitive to methyl guanine methyltransferase treatment, indicating that the mutations are formed on O⁶-methylguanine modifications. Human Pol η reduced the SBS11-like mutations, indicating its suppressive role in TMZ-induced mutagenesis. Yeast Pol ζ and human Pol κ generated distinct mutations unrelated to SBS11.