AID is related to enzymes that deaminate cytosine to uracil in making nucleotide precursors for RNA and DNA synthesis. Its closest homolog, APOBEC1 (apolipoprotein B mRNA editing catalytic polypeptide 1), is an RNA-editing enzyme that deaminates cytosine in the context of RNA. However, AID fulfills its activity in antibody gene diversification by acting on cytosine in the DNA of the immunoglobulin locus. When AID deaminates cytidine residues in the immunoglobulin V regions, somatic hypermutation is initiated; when cytidine residues in switch regions are deaminated, class switch recombination is initiated.
AID can deaminate cytidine residues in single-stranded DNA but not double-stranded DNA (Fig. 10.18). For AID to act, AID target genes are typically being transcribed, so that the DNA double helix is temporarily unwound. Since AID is expressed only in germinal center B cells, targeting of the immunoglobulin genes takes place only in these cells and in the actively transcribed rearranged
The activity of AID, which is expressed only in B cells, requires access to the cytidine side chain of a single-stranded DNA molecule (first panel), which is normally prevented by the hydrogen bonding in double-stranded DNA. AID initiates a nucleophilic attack on the exposed cytosine ring (second panel), which is resolved by the deamination of the cytidine to form uridine (third panel).
Fig. 10.19 AID initiates DNA lesions whose repair leads to somatic hypermutation, class switch recombination, or gene conversion.
When AID converts a cytidine (C) to uridine (U) in the DNA of an immunoglobulin gene, the final mutation produced depends on which repair pathways are used. Somatic hypermutation can result from either the mismatch repair (MSH2/6) pathway combined with error-prone polymerase activity of Polη, or the base-excision repair (UNG) pathway. Acting together, these can generate point mutations at and around the site of the original C:G pair. REV1 is a DNA repair enzyme that can synthesize DNA, or recruit other enzymes that can synthesize DNA, over the abasic sites in damaged DNA. REV1 itself will insert only C opposite the abasic site, but it can help recruit other polymerases that can also insert A, G, and T. The end result is insertion of a random nucleotide at the C:G residues where AID initially acted. Both class switch recombination and gene conversion require the formation of a single-strand break in the DNA. A single-strand break is formed when apurinic/apyrimidinic endonuclease 1 (APE1) removes a damaged residue from the DNA as part of the repair process (see Fig. 10.20, bottom two panels). In class switch recombination, single-strand breaks made in two of the so-called switch regions upstream of the C-region genes are converted to double-strand breaks. The cell’s machinery for repairing double-strand breaks, which is very similar to the later stages of V(D)J recombination, then rejoins the DNA ends in a way that leads to a recombination event in which a different C-region gene is brought adjacent to the rearranged V region. Gene conversion results from the broken DNA strand using homologous sequences flanking the immunoglobulin gene as a template for repair DNA synthesis, thus replacing part of the gene with new sequences.
V regions where RNA polymerase generates transient single-stranded regions. Somatic hypermutation does not occur in loci that are not being actively transcribed. Rearranged VH and VL genes are mutated even if they are ‘nonproductive’ rearrangements and are not being expressed as protein, as long as they are being transcribed. Some actively transcribed genes in B cells besides those for immunoglobulins can also be affected by the somatic mutation process, but at a much lower rate.