Matthew D. Scharff

The expression of activation-induced cytidine deaminase (AID) is prerequisite to a "trifecta" of key molecular events in B cells: class-switch recombination and somatic hypermutation in humans and mice and gene conversion in chickens. Although this critically important enzyme shares common sequence motifs with apolipoprotein B mRNA-editing enzyme, and exhibits deaminase activity on free deoxycytidine in solution, it has not been shown to act on either RNA or DNA. Recent mutagenesis data in Escherichia coli suggest that AID may deaminate dC on DNA, but its putative biochemical activities on either DNA or RNA remained a mystery. Here, we show that AID catalyzes deamination of dC residues on single-stranded DNA in vitro but not on double-stranded DNA, RNA-DNA hybrids, or RNA. Remarkably, it has no measurable deaminase activity on single-stranded DNA unless pretreated with RNase to remove inhibitory RNA bound to AID. AID catalyzes dC --> dU deamination activity most avidly on double-stranded DNA substrates containing a small "transcription-like" single-stranded DNA bubble, suggesting a targeting mechanism for this enigmatic enzyme during somatic hypermutation.

Affinity maturation of the humoral response is mediated by somatic hypermutation of the immunoglobulin (Ig) genes and selection of higher-affinity B cell clones. Activation-induced cytidine deaminase (AID) is the first of a complex series of proteins that introduce these point mutations into variable regions of the Ig genes. AID deaminates deoxycytidine residues in single-stranded DNA to deoxyuridines, which are then processed by DNA replication, base excision repair (BER), or mismatch repair (MMR). In germinal center B cells, MMR, BER, and other factors are diverted from their normal roles in preserving genomic integrity to increase diversity within the Ig locus. Both AID and these components of an emerging error-prone mutasome are regulated on many levels by complex mechanisms that are only beginning to be elucidated.

The S107 IgA kappa-chain myeloma cell line makes an antiphosphocholine antibody of the T15 idiotype. A somatic mutant of this line, U4, makes an immunoglobulin with a single amino acid substitution of an alanine for a glutamic acid at residue 35. This single amino acid change results in a loss of phosphocholine binding activity. However, the U4 immunoglobulin has acquired reactivity with a variety of phosphorylated macromolecules, including double-stranded DNA, protamine, and cardiolipin. Thus, a single amino acid change in the T15 heavy chain can transform an antibacterial antibody into an antibody that resembles the autoantibodies seen in mice and man with autoimmune disease.