AID mutates a non-immunoglobulin transgene independent of chromosomal position.

It is unknown how activation-induced cytidine deaminase (AID) targets immunoglobulin (Ig) genes during somatic hypermutation. Results to date are difficult to interpret: while some results argue that Ig genes have special sequences that mobilize AID, other work shows that non-Ig transgenes mutate. In this report, we have examined the effects of the intronic mu enhancer on the somatic hypermutation rates of a retroviral vector. For this analysis, we used centroblast-like Ramos cells to capture as much of the natural process as possible, used AIDhi and AIDlow Ramos variants to ensure that mutations are AID induced, and measured mutation of a GFP-provirus to achieve greater sensitivity. We found that mutation rates of the non-Ig provirus were AID-dependent, were similar at different genomic loci, but were approximately 10-fold lower than the V-region suggesting that AID can mutate non-Ig genes at low rates. However, the intronic mu enhancer did not increase the mutation rates of the provirus. Interestingly, exogenous over-expression of AID revealed that the V-region mutation rate can be saturated by lower levels of AID than the provirus, suggesting that selective mutation of Ig sequences is compromised in cells that over-express AID.

Antibody diversification: mutational mechanisms and oncogenesis.

Antibody diversification processes play a major role in protecting humans from pathogens. Somatic hypermutation and gene conversion increase the affinity of pathogen-specific antibodies by changing the sequence within antibody variable genes, while the class switch recombination (CSR) process changes the antibody's effector function by replacing the constant region of the antibody gene with a different constant region. Activation-induced cytidine deaminase (AID) initiates each of these three processes by deaminating cytidines within antibody genes, while a host of other DNA transacting factors are involved in either creating new mutations or repairing DNA lesions introduced during these processes. This review will discuss the main features of antibody diversification and their role in lymphomagenesis, highlight outstanding issues and questions that remain in the field, and discuss our contributions to this field.

The mutation spectrum of purified AID is similar to the mutability index in Ramos cells and in ung(-/-)msh2(-/-) mice.

Somatic hypermutation and class switch recombination are initiated by the enzyme activation-induced cytidine deaminase (AID). Although other models exist for AID function, one model suggests that AID initiates these processes by deaminating cytidines within DNA, thereby initiating mutagenic repair pathways that involve either UNG or Msh2. Recent work shows that GST-hAID prefers to mutate WRC motifs, a motif frequently mutated in vivo. Because this is a strong argument in favor of the DNA deamination model, we sought to extend this analysis by examining the activity of purified AID with a small polyhistidine tag (His-hAID) on all 16 trinucleotide combinations (i.e., NNC). Here we show that purified His-hAID preferentially mutated cytidines within WRC (i.e., A/T, A/G, C) motifs, but poorly mutated cytidines within GYC (G, C/T, C) motifs. We next compared this mutability preference with those in hypermutating Ramos cells and in msh2(-/-)ung(-/-) mice, since both are reduced or deficient in UNG- and/or Msh2-induced mutations and are thus likely to reflect the sequence specificity of the mutator in vivo. Indeed, the mutation spectrums of purified His-hAID and GST-hAID matched the trinucleotide mutability indexes in Ramos cells and in msh2(-/-)ung(-/-) mice. Thus, the activity of AID on single-stranded DNA produces the same mutation pattern as double-stranded DNA in hypermutating cells. These data lend support to the DNA deamination model and indicate that AID does not require co-factors for its WRC specificity.