A study published recently in Molecular Cell provides new insight into the molecular mechanisms of antibody diversification through class-switch recombination (CSR) and somatic hypermutation (SHM) downstream of activation-induced cytidine deaminase (AID), which is required for both processes.

The most widely accepted model of the mechanism of CSR (see Further reading) is that AID deaminates cytosine (C) residues to uracil (U) residues, yielding U·guanine (G) mismatched pairs, and that this is followed by excision of the mismatched uracil residue by uracil-DNA glycosylase (UNG). However, although UNG deficiency severely impairs CSR, it does not completely abolish CSR in vivo.

Similar uncertainty exists about the mechanism of SHM. Neuberger and colleagues have previously proposed two phases of SHM, the first focused on C·G pairs and the second on adenine (A)·thymine (T) pairs. They suggest that AID-generated U·G pairs lead to C·G mutations and that mutations at A·T pairs arise as a result of recognition of the initial AID-generated U·G pair by mismatch-repair proteins (such as mutS homologue 2, MSH2) and error-prone polymerases. However, although deficiency in such proteins reduces A·T mutations, they are not completely abolished.

The authors therefore set out to characterize these back-up pathways of CSR and SHM. Although it has been suggested that SMUG1 (single-strand selective monofunctional uracil-DNA glycosylase 1) — which has been shown in biochemical assays to mediate uracil excision in UNG-deficient tissue extracts — could mediate the UNG-independent pathway of CSR, B cells from UNG-deficient mice that are transgenic for human SMUG1 remained impaired in their ability to mediate CSR in vitro. By contrast, B cells from mice lacking both MSH2 and UNG were unable to switch in vitro or in vivo, as shown by a lack of B220+IgG1+ cells after culture in the presence of both lipopolysaccharide and interleukin-4 and by the barely detectable levels of IgG and IgA in the serum of these mice, respectively.

Further analysis of the sequence of the 3′-intronic flank of rearranged VHDJH genes in Peyer's patch germinal-centre B cells from mice deficient in MSH2 and UNG indicated that SHM A·T mutations were eliminated in the double-knockout animals. In addition, mutations at C·G pairs were restricted to C·G to T·A transitions, rather than the usual combination of transitions and transversions (by which C·G mutations result in either A·T or G·C). This indicates that all of the mutations occur as a result of replication over the U·G mismatch and provides evidence that either mismatch recognition by MSH2 or base excision by UNG is required for A·T mutations in the second phase of SHM. A similarly restricted pattern of C·G to T·A transition mutations was observed at the IgM switch region of germinal-centre B cells from mice deficient in MSH2 and UNG, supporting the conclusion that MSH2- and UNG-mediated processes are the two pathways that resolve the AID-generated U·G mismatches during CSR.

This study furthers our understanding of the molecular mechanisms downstream of AID that result in CSR and the second phase of SHM (that is, the introduction of mutations at A·T pairs): mismatch recognition by MSH2 backs up UNG-mediated uracil excision for CSR, whereas the reverse is true of mutations in phase two of SHM. Future studies will define further specific molecular requirements for these two processes.