Somatic Hypermutation and Class Switch Recombination in Msh6−/−Ung−/− Double-Knockout Mice

Abstract

Somatic hypermutation (SHM) and class switch recombination (CSR) are initiated by activation-induced cytosine deaminase (AID). The uracil, and potentially neighboring bases, are processed by error-prone base excision repair and mismatch repair. Deficiencies in Ung, Msh2, or Msh6 affect SHM and CSR. To determine whether Msh2/Msh6 complexes which recognize single-base mismatches and loops were the only mismatch-recognition complexes required for SHM and CSR, we analyzed these processes in Msh6−/−Ung−/− mice. SHM and CSR were affected in the same degree and fashion as in Msh2−/−Ung−/− mice; mutations were mostly C,G transitions and CSR was greatly reduced, making Msh2/Msh3 contributions unlikely. Inactivating Ung alone reduced mutations from A and T, suggesting that, depending on the DNA sequence, varying proportions of A,T mutations arise by error-prone long-patch base excision repair. Further, in Msh6−/−Ung−/− mice the 5′ end and the 3′ region of Ig genes was spared from mutations as in wild-type mice, confirming that AID does not act in these regions. Finally, because in the absence of both Ung and Msh6, transition mutations from C and G likely are “footprints” of AID, the data show that the activity of AID is restricted drastically in vivo compared with AID in cell-free assays.

DNA polymerases in adaptive immunity

“…4). By contrast, MSH2 deficiency results in a decreased mutagenesis at A/T bases, a phenotype that is also associated with MSH6 and Exo1 deficiency [100][101][102][103] (but which is not observed in the absence of PMS2 or MLH1, the effector partners of the mismatch repair complex) 42,104 . These contrasting phenotypes led to the proposition of two alternative phases of SHM: one mediated by UNG and generating mutations at G/C bases after uracil excision, and one mediated by MSH2-MSH6, introducing A/T mutations 98 .…”

Separation of Function between Isotype Switching and Affinity Maturation In Vivo during Acute Immune Responses and Circulating Autoantibodies in UNG-Deficient Mice

“…UNG and MSH2/ MSH6 are partially redundant for SHM and CSR, but they are not completely equivalent. In vitro CSR to IgG1 and IgG3 (i.e., measured in purified, cytokine-stimulated naive B cells from mice) is reduced by .10-fold in Ung 2/2 B cells, but only by ∼3-fold in MSH2/MSH6-deficient B cells (9)(10)(11)(12)(13)(14)(15). However, Msh2 2/2 mice produce up to 10-fold less anti-NP IgG1 titers than wild-type (wt) mice after immunization (15,16).…”

“…However, Msh2 2/2 mice produce up to 10-fold less anti-NP IgG1 titers than wild-type (wt) mice after immunization (15,16). Furthermore, Msh2 2/2 and Msh6 2/2 mice show not only severe skewing of the SHM spectrum because of reduced SHM at A:T pairs, but also a change in the distribution of mutations and significant reduction in overall SHM accumulation (11,13,16,17). The defects on Ab diversification displayed by Msh2 2/2 mice are therefore more severe than what could be predicted from the defects measured by in vitro assays.…”

“…1A). Indeed, the simultaneous absence of both UNG and MSH2/MSH6 results in SHM restricted to C:G-to-T:A mutations and the complete absence of all secondary isotypes and only IgM circulating in the serum of these mice (10,11). UNG and MSH2/ MSH6 are partially redundant for SHM and CSR, but they are not completely equivalent.…”

“…Moreover, preimmune serum Ig levels could be confounded by homeostatic compensation. The analysis of Ab responses to experimental immunization in Ung 2/2 mice is limited to measuring the primary response against SRBC, which was significantly but quite modestly reduced for some isotypes (11). Thus, it is, in fact, still uncertain how much MSH2/MSH6 could compensate for UNG in catalyzing CSR in vivo after immunization or infection, and whether it may do so differently for different Ig classes.…”