APOBEC3A and APOBEC3B Preferentially Deaminate the Lagging Strand Template during DNA Replication.

APOBEC family cytidine deaminases have recently been implicated as powerful mutators of cancer genomes. How APOBECs, which are ssDNA-specific enzymes, gain access to chromosomal DNA is unclear. To ascertain the chromosomal ssDNA substrates of the APOBECs, we expressed APOBEC3A and APOBEC3B, the two most probable APOBECs mediating cancer mutagenesis, in a yeast model system. We demonstrate, using mutation reporters and whole genome sequencing, that APOBEC3A- and APOBEC3B-induced mutagenesis primarily results from the deamination of the lagging strand template during DNA replication. Moreover, our results indicate that both genetic deficiencies in replication fork-stabilizing proteins and chemical induction of replication stress greatly augment the mutagenesis of APOBEC3A and APOBEC3B. Taken together, these results strongly indicate that ssDNA formed during DNA lagging strand synthesis is a major substrate for APOBECs and may be the principal substrate in human cancers experiencing replication stress.