SRP372487 PRJNA832427 Mutational impact of APOBEC3B and APOBEC3A in a human cell line A prominent source of mutation in cancer is single-stranded DNA cytosine deamination by cellular APOBEC3 enzymes, which results in C to T and C to G mutations in TCA and TCT motifs. Although multiple enzymes have been implicated, reports conflict and it is unclear which enzyme(s) are responsible. Here we develop a selectable system to quantify genome mutation and compare the mutagenic activities of three leading candidates APOBEC3A, APOBEC3B, and APOBEC3H. The human cell line, HAP1, was engineered to express the thymidine kinase (TK) gene of HSV1, which confers sensitivity to ganciclovir. Clonal expression of APOBEC3A and APOBEC3B, but not catalytic mutant controls or APOBEC3H, triggered elevated DNA damage responses and increased frequencies of TK mutation. Mutant TK DNA sequences revealed nearly indistinguishable cytosine mutation patterns. Whole genome sequences from TK mutant clones confirmed these results and enabled broader bioinformatic analyses. Most importantly, comparisons of pure APOBEC3A and APOBEC3B inflicted mutation signatures from this system and the actual APOBEC3 signature from multiple different cancer types indicated that most tumors manifest a composite signature. These studies help resolve a long-standing etiologic debate in the cancer field and indicate that future diagnostic and therapeutic efforts should focus on these two enzymes.