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The APOBEC3 family of cytosine deaminases has been implicated in some of the most prevalent mutational signatures in cancer1-3. However, a causal link between endogenous APOBEC3 enzymes and mutational signatures in human cancer genomes has not been established, leaving the mechanisms of APOBEC3 mutagenesis poorly understood. Here, to investigate the mechanisms ofAPOBEC3 mutagenesis, we deleted implicated genes from human cancer cell lines that naturally generate APOBEC3-associated mutational signatures over time4. Analysis of non-clustered and clustered signatures across whole-genome sequences from 251 breast, bladder and lymphoma cancer cell line clones revealed that APOBEC3A deletion diminished APOBEC3-associated mutational signatures. Deletion of both APOBEC3A and APOBEC3B further decreased APOBEC3 mutation burdens, without eliminating them. Deletion of APOBEC3B increased APOBEC3A protein levels, activity and APOBEC3A-mediated mutagenesis in some cell lines. The uracil glycosylase UNG was required for APOBEC3-mediated transversions, whereas the loss ofthe translesion polymerase REV1 decreased overall mutation burdens. Together, these data represent direct evidence that endogenous APOBEC3 deaminases generate prevalent mutational signatures in human cancer cells. Our results identify APOBEC3A as the main driver of these mutations, indicate that APOBEC3B can restrain APOBEC3A-dependent mutagenesis while contributing its own smaller mutation burdens and dissect mechanisms that translate APOBEC3 activities into distinct mutational signatures.
Early investigations into the patterns of mutations in cancer genomes revealed that cytosine mutations are commonly present in TCN (where N is any nucleotide) trinucleotide sequence contexts1,2,5. The sequence context preferences of the APOBEC cytosine deaminases, which target DNA and RNA ofviruses and retroelements as part of the innate immune defence, led to the proposal that such mutations derive from APOBEC activity1-3,6,7. Mathematical deconvolution of patterns of single-base substitutions (SBSs) from cancer genomes uncovered different mutational signatures of non-clustered (termed signatures SBS2 and SBS13) and clustered (kataegis and omikli) APOBEC-associated cytosine mutations at TCN trinucleotides1,8,9. APOBEC-associated mutational signatures have been identified in more than 70% of cancer types and around 50% of all cancer genomes, with prominence in breast and bladder cancer as well as other cancer types10,11. Indirect links implicate the APOBEC3 family as a source of these mutations: (1) APOBEC3 overexpression in model systems produces cytosine mutations with features that are similar to SBS2 and SBS13 and can contribute to carcinogenesis; (2) polymorphisms at the APOBEC3 locus are, in some contexts, associated...