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Activation-induced deaminase (AID) uses base deamination for class-switch recombination and somatic hypermutation and is related to the mammalian RNA-editing enzyme apolipoprotein B editing catalytic subunit 1 (APOBEC-1). CDD1 is a yeast ortholog of APOBEC-1 that exhibits cytidine deaminase and RNA-editing activity. Here, we present the crystal structure of CDD1 at 2.0-Å resolution and its use in comparative modeling of APOBEC-1 and AID. The models explain dimerization and the need for trans-acting loops that contribute to active site formation. Substrate selectivity appears to be regulated by a central active site ''flap'' whose size and flexibility accommodate large substrates in contrast to deaminases of pyrimidine metabolism that bind only small nucleosides or free bases. Most importantly, the results suggested both AID and APOBEC-1 are equally likely to bind single-stranded DNA or RNA, which has implications for the identification of natural AID targets.A ntibody diversity is generated during B cell development through class-switch recombination (CSR) and somatic hypermutation (SHM) (1), and in many vertebrates by gene conversion (2). Expression of activation-induced deaminase (AID) is critical for all three processes (3-5) and ectopic AID expression was sufficient to activate SHM in B cell lines, hybridomas, and fibroblasts (6-9), gene conversion in B cells (4, 5), as well as CSR in fibroblast cell lines (10). Mutations in the AID protein have been detected in humans with hyper-IgM type 2 syndrome (11). Expression of AID in Escherichia coli caused deoxycytidine-todeoxyuridine deamination in actively transcribed host genes that was enhanced in strains deficient in the deoxyuridine repair enzyme DNA uracil N-glycosylase (12). DNA uracil N-glycosylase deficiency in mammals resulted in altered patterns of CSR and SHM (13,14). In vitro studies revealed that AID catalyzed deamination on single-stranded DNA at WRCY hot spots, but not on doublestranded DNA, RNA-DNA hybrids, or single-stranded RNA (15,16). Cytidine deamination on single-stranded DNA within transcription bubbles (17, 18) and the observation that transcription rates influenced SHM activity (17)(18)(19) suggested that the biological substrate for AID is DNA.In contrast, the homology of AID to the mRNA-editing enzyme APOBEC-1 (3), suggested AID might edit RNA (3). This idea was intriguing because edited mRNAs either encode novel proteins or lose the ability to express proteins (20,21). Consistent with expression of a novel protein from edited mRNA, de novo protein synthesis was required for CSR subsequent to AID activation (22).APOBEC-1 and AID have proven difficult to purify at levels sufficient for structural studies. CDD1 from Saccharomyces cerevisiae (ScCDD1) can be purified readily, which is relevant due to its orthology with APOBEC-1 at the level of both cytidine deaminase (CDA) sequence similarity (27%) and mRNA-editing activity on apolipoprotein B (apoB) substrates (23). We determined the crystal structure of ScCDD1 to 2.0 Å resolution, which ...

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