Mutational impact of APOBEC3B and APOBEC3A in a human cell line
Introduction
Over the past decade, the APOBEC3 family of single-stranded (ss)DNA cytosine deaminases has emerged as one of the top three sources of single base substitution (SBS) mutation in cancer with particularly large contributions to tumors of the bladder, breast, cervix, lung, and head/neck. The deamination of single-stranded DNA cytosines by cellular APOBEC3 enzymes, leading to C-to-T and C-to-G mutations in TCA and TCT motifs. Although multiple enzymes have been implicated, there is conflicting evidence regarding which enzyme(s) are responsible.
Methods
We developed a selectable system to quantify genome mutations and compared the mutagenic activities of three primary candidates - APOBEC3A, APOBEC3B, and APOBEC3H. We engineered the human HAP1 cell line to express the herpes simplex virus-1 thymidine kinase (TK) gene, conferring sensitivity to ganciclovir.
Results
Expression of APOBEC3A in three distinct human cell lines resulted in increased DNA damage 24 hours post-transfection. Clonal expression of APOBEC3A and APOBEC3B, but not catalytic mutant controls or APOBEC3H, led to an increase in TK mutation frequency. DNA sequences from mutant TKs revealed almost identical cytosine mutation patterns. Whole genome sequences from TK mutant clones confirmed these findings, providing a foundation for more extensive bioinformatic analyses. Most importantly, the comparison of mutation signatures inflicted by APOBEC3A and APOBEC3B in our system, with the actual APOBEC3 signature from breast cancer, suggests that most tumors likely exhibit a composite signature.
Conclusion
Here we report the development and implementation of a novel system to investigate mutational processes in human cells. The studies here demonstrate unambiguously that both APOBEC3A and APOBEC3B can inflict an APOBEC3 mutation signature in human genomic DNA with, in both instances, ssDNA deamination events immortalizing predominantly as C-to-T and C-to-G mutations in TCA and TCT trinucleotide motifs. These studies help clarify a long-standing debate in cancer etiology and suggest that future diagnostic and therapeutic strategies should target both APOBEC3A and APOBEC3B.