APOBEC cytidine deaminases are the second-most prominent source of mutagenesis in sequenced tumors. Previous studies have proposed that APOBEC3B (A3B) is the major source of mutagenesis in breast cancer (BRCA). We show that APOBEC3A (A3A) is the only APOBEC whose expression correlates with APOBEC-induced mutation load and that A3A expression is responsible for cytidine deamination in multiple BRCA cell lines. Comparative analysis of A3A and A3B expression by qRT-PCR, RSEM-normalized RNA-seq, and unambiguous RNA-seq validated the use of RNA-seq to measure APOBEC expression, which indicates that A3A is the primary correlate with APOBEC-mutation load in primary BRCA tumors. We also demonstrate that A3A has >100-fold more cytidine deamination activity than A3B in the presence of cellular RNA, likely explaining why higher levels of A3B expression contributes less to mutagenesis in BRCA. Our findings identify A3A as a major source of cytidine deaminase activity in breast cancer cells and possibly a prominent contributor to the APOBEC mutation signature.

The cytidine deaminase, APOBEC3A (A3A), is a prominent source of mutations in multiple cancer types. These APOBEC-signature mutations are non-uniformly distributed across cancer genomes, associating with single-stranded (ss) DNA formed during DNA replication and hairpin-forming sequences. The biochemical and cellular factors that influence these specificities are unclear. We measured A3A’s cytidine deaminase activity in vitro on substrates that model potential sources of ssDNA in the cell and found that A3A is more active on hairpins containing 4 nt ssDNA loops compared to hairpins with larger loops, bubble structures, replication fork mimics, ssDNA gaps, or linear DNA. Despite pre-bent ssDNAs being expected to fit better in the A3A active site, we determined A3A favors a 4 nt hairpin substrate only 2- to fivefold over linear ssDNA substrates. Addition of whole cell lysates or purified RPA to cytidine deaminase assays more severely reduced A3A activity on linear ssDNA (45 nt) compared to hairpin substrates. These results indicate that the large enrichment of A3A-driven mutations in hairpin-forming sequences in tumor genomes is likely driven in part by other proteins that preferentially bind longer ssDNA regions, which limit A3A’s access. Furthermore, A3A activity is reduced at ssDNA associated with a stalled T7 RNA polymerase, suggesting that potential protein occlusion by RNA polymerase also limits A3A activity. These results help explain the small transcriptional strand bias for APOBEC mutation signatures in cancer genomes and the general targeting of hairpin-forming sequences in the lagging strand template during DNA replication.

The initiation, progression, and relapse of cancers often result from mutations occurring within somatic cells. Consequently, processes that elevate mutation rates accelerate carcinogenesis and hinder the development of long-lasting therapeutics. Recent sequencing of human cancer genomes has identified patterns of mutations, termed mutation signatures, many of which correspond to specific environmentally induced and endogenous mutation processes. Some of the most frequently observed mutation signatures are caused by dysregulated activity of APOBECs, which deaminate cytidines in single-stranded DNA at specific sequence motifs causing C-to-T and C-to-G substitutions. In humans, APOBEC-generated genetic heterogeneity in tumor cells contributes to carcinogenesis, metastasis, and resistance to therapeutics. Here, we review the current understanding of APOBECs’ role in cancer mutagenesis and impact on disease and the biological processes that influence APOBEC mutagenic capacity. Expected final online publication date for the Annual Review of Genetics, Volume 56 is November 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.