Mutation signatures specific to DNA alkylating agents in yeast and cancers

Alkylation is one of the most ubiquitous forms of DNA lesions. However, the motif preferences and substrates for the activity of the major types of alkylating agents defined by their nucleophilic substitution reactions (S(N)1 and S(N)2) are still unclear. Utilizing yeast strains engineered for large-scale production of single-stranded DNA (ssDNA), we probed the substrate specificity, mutation spectra and signatures associated with DNA alkylating agents. We determined that S(N)1-type agents preferably mutagenize double-stranded DNA (dsDNA), and the mutation signature characteristic of the activity of S(N)1-type agents was conserved across yeast, mice and human cancers. Conversely, S(N)2-type agents preferably mutagenize ssDNA in yeast. Moreover, the spectra and signatures derived from yeast were detectable in lung cancers, head and neck cancers and tumors from patients exposed to S(N)2-type alkylating chemicals. The estimates of mutation loads associated with the S(N)2-type alkylation signature were higher in lung tumors from smokers than never-smokers, pointing toward the mutagenic activity of the S(N)2-type alkylating carcinogens in cigarettes. In summary, our analysis of mutations in yeast strains treated with alkylating agents, as well as in whole-exome and whole-genome-sequenced tumors identified signatures highly specific to alkylation mutagenesis and indicate the pervasive nature of alkylation-induced mutagenesis in cancers.