Discovery of cytosine deaminases enables base-resolution methylome mapping using a single enzyme.

Deaminases have important uses in modification detection and genome editing. However, the range of applications is limited by the small number of characterized enzymes. To expand the toolkit of deaminases, we developed an in vitro approach that bypasses a major hurdle with their toxicity in cells. We assayed 175 putative cytosine deaminases on a variety of substrates and found a broad range of activity on double- and single-stranded DNA in various sequence contexts, including CpG-specific deaminases and enzymes without sequence preference. We also characterized enzyme selectivity across six DNA modifications and reported enzymes that do not deaminate modified cytosines. The detailed analysis of diverse deaminases opens new avenues for biotechnological and medical applications. As a demonstration, we developed SEM-seq, a non-destructive single-enzyme methylation sequencing method using a modification-sensitive double-stranded DNA deaminase. The streamlined protocol enables accurate, base-resolution methylome mapping of scarce biological material, including cell-free DNA and 10 pg input DNA.

APOBECs orchestrate genomic and epigenomic editing across health and disease.

APOBEC proteins can deaminate cytosine residues in DNA and RNA. This can lead to somatic mutations, DNA breaks, RNA modifications, or DNA demethylation in a selective manner. APOBECs function in various cellular compartments and recognize different nucleic acid motifs and structures. They orchestrate a wide array of genomic and epigenomic modifications, thereby affecting various cellular functions positively or negatively, including immune editing, viral and retroelement restriction, DNA damage responses, DNA demethylation, gene expression, and tissue homeostasis. Furthermore, the cumulative increase in genomic and epigenomic editing with aging could also, at least in part, be attributed to APOBEC function. We synthesize our cumulative understanding of APOBEC activity in a unifying overview and discuss their genomic and epigenomic impact in physiological, pathological, and technological contexts.

APOBECs and virus restriction.

The APOBEC family of single-stranded DNA cytosine deaminases comprises a formidable arm of the vertebrate innate immune system. Pre-vertebrates express a single APOBEC, whereas some mammals produce as many as 11 enzymes. The APOBEC3 subfamily displays both copy number variation and polymorphisms, consistent with ongoing pathogenic pressures. These enzymes restrict the replication of many DNA-based parasites, such as exogenous viruses and endogenous transposable elements. APOBEC1 and activation-induced cytosine deaminase (AID) have specialized functions in RNA editing and antibody gene diversification, respectively, whereas APOBEC2 and APOBEC4 appear to have different functions. Nevertheless, the APOBEC family protects against both periodic viral zoonoses as well as exogenous and endogenous parasite replication. This review highlights viral pathogens that are restricted by APOBEC enzymes, but manage to escape through unique mechanisms. The sensitivity of viruses that lack counterdefense measures highlights the need to develop APOBEC-enabling small molecules as a new class of anti-viral drugs.