Abstract
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.
Highlights
APOBEC3 (“apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like”-3) family enzymes are cytidine deaminases that function as innate immune response factors through their binding and deamination of viruses and transposons, which repress the replication of these elements[1,2,3]
Cytidine deaminase activity at the TTCA sequence in each substrate creates a deoxyuridine base, which is converted to a heat-labile abasic site by the activity of uracil-DNA glycosylase (UDG), resulting in shorter single-stranded DNA (ssDNA) fragments (Fig. 1B) that are resolved from uncleaved substrate on a denaturing polyacrylamide gel
The sequence of our hairpin substrate consists of a hotspot for APOBEC mutagenesis in breast cancers[25] likely due to A3A’s affinity for U-shaped ssDNA present in hairpin s tructures[30,49,50]
Summary
APOBEC3 (“apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like”-3) family enzymes are cytidine deaminases that function as innate immune response factors through their binding and deamination of viruses and transposons, which repress the replication of these elements[1,2,3]. Biochemical studies of substrate preferences have been instrumental in understanding the targeting of mutations caused by the related APOBEC, AID, to transcription intermediates formed in immunoglobulin loci These efforts initially highlighted the structural loops required for AID WRC sequence s pecificity[33], determined that AID (and by extension most other APOBECs) is ssDNA specific[34,35,36], and identified roles for transcription[37], R-loops[38], G-quadruplexes39, RPA40, and RNA exosome complex activity in allowing AID to deaminate both strands of a transcription unit[41]. We determined that A3A deamination activity is low on ssDNA bubble substrates, like those found during transcription, using an in vitro transcription assay These results indicate that DNA structure and competition with other ssDNA binding proteins are important factors in predicting at-risk locations in the human genome for A3A-induced mutagenesis
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