Biochemical analysis of APOBEC3A-induced mutational signatures and clustered mutagenesis.

APOBEC3A is a single-stranded DNA cytidine deaminase that is involved in mutagenic processes in various cancers. Ample evidence indicates that APOBEC3A causes two major mutational signatures (SBS2 and SBS13) in cancer cells and cellular model systems, but the detailed molecular mechanisms that generate these two signatures from a single deaminase remain unclear. APOBEC3A-induced mutations are preferentially observed at DNA hairpin structures and are often observed as clusters in cancer genomes, but the molecular mechanisms behind the clustered mutations are unclear. Here, we developed a cell-free system to analyze the mutations on APOBEC3A-treated DNA and successfully reproduced SBS2 and a portion of SBS13 by using specific combinations of DNA polymerases. While SBS2 can be produced by a replicative DNA polymerase, SBS13 requires the activities of uracil DNA glycosylase, Rev1, and Pol ζ. We also found that the DNA hairpin structures have positive and negative influences on deaminase activity depending on the position of cytosine residues within the loop. In our system, APOBEC3A-induced mutagenesis showed positive cooperativity by which one mutation stimulated a second mutation at up to 50-nt distance. This cooperative effect promoted clustered mutagenesis in vitro, but little continuity of mutations was observed among the multi-mutation products. These observations support an aggregation-mediated mechanism of clustered mutagenesis.