Hypomorphic mutation in the large subunit of replication protein A affects mutagenesis by human APOBEC cytidine deaminases in yeast

Human APOBEC single-strand (ss)-specific DNA and RNA cytidine deaminases function in RNA editing and in antiviral innate immunity but can also cause mutagenesis in chromosomes by changing cytosines to uracils. C to T mutations can arise from direct replication of uracils. Alternatively, abasic (AP) sites created by the uracil DNA glycosylase can be fixed as C to T or C to G mutations by translesion DNA polymerases. We noticed that in yeast and in human cancers contributions of C to T and C to G mutations depends on the origin of ssDNA mutagenized by APOBECs. Since ssDNA in eukaryotic genomes readily binds to replication protein A (RPA) we asked if RPA could affect APOBEC-induced mutation spectrum in yeast. For that purpose, we expressed human APOBECs in the wild-type yeast and in strains carrying a hypomorph mutation rfa1-t33 in the large RPA subunit. We confirmed that the rfa1-t33 allele can facilitate mutagenesis by APOBECs. We also found that rfa1-t33 mutation shifted the ratio of APOBEC3A-induced T to C and T to G mutations in replicating yeast to resemble a ratio observed in long persistent ssDNA in yeast and in cancers. We present the data suggesting that RPA may shield uracils formed by APOBECs in ssDNA from uracil DNA glycosylase Ung1, thereby facilitating C to T mutagenesis through accurate copying of uracils by replicative DNA polymerases. Unexpectedly, we also found that for uracils shielded from Ung1 by wild-type RPA the mutagenic outcome is reduced in the presence of translesion DNA polymerase zeta.