The mismatch repair pathway functions normally at a non-AID target in germinal center B cells

Abstract

IntroductionMismatch repair (MMR) is an evolutionary conserved DNA repair pathway that is required to repair mutations that arise through DNA replication or other processes. 1,2 MutS homologue 2 (Msh2) dimerizes with either Msh6 or Msh3 to recognize single base-pair mismatches or mismatches caused by insertion/deletions, respectively. 2 After binding to the mismatch, Msh2 hydrolyzes ATP, which induces a change in the heterodimer that allows for the recruitment of downstream repair factors such as MutL homologue 1, postmeiotic segregation 2, and exonuclease 1 (Exo1). 3,4 Because of Msh2's essential role in repairing DNA mutations, Msh2 Ϫ/Ϫ mice have 5-fold, 11-fold, and 15-fold greater mutation frequencies in the brain, small intestine, and thymus, respectively, 5 and are predisposed to cancer. [6][7][8] During an immune response, Ig genes undergo somatic hypermutation (SHM) to produce high-affinity antibodies. SHM is initiated by activation-induced cytidine deaminase (AID), 9 which deaminates cytidine molecules within the V region to produce dU:dG mispairs. [10][11][12] The dU:dG mismatch is recognized by the Msh2/Msh6 heterodimer, but instead of repairing this mutation, MMR produces mutations. [13][14][15][16][17][18] Mice deficient in Msh2, Msh6, postmeiotic segregation 2, MutL homologue 1, or Exo1 have an approximately 2-fold decrease in overall mutation frequency at the V region. 13,14,[16][17][18] In particular, there is an approximately10-fold decrease in mutations at A:T base pairs at the V region in MSH2-deficient mice, whereas the mutation frequency at G:C base pairs is unaltered. [15][16][17][18][19] These results indicate that the MMR pathway is required to produce mutations at A:T base pairs in the V region. MMR proteins have also been implicated in mutagenic processes that cause disease, including trinucleotide-repeat diseases. 20 Two sets of recent results suggest the possibility that the MMR system is generally defective in germinal center (GC) B cells.Ouchida et al 21 reported that GC B cells have a generally greater mutation rate than other cells, as measured by the frequency of mutations on a lacZ transgene. Liu et al 22 found that in Msh2 mutant mice, the mutation frequency of AID-sensitive genes was either lower or only slightly greater than in wild-type (WT) cells; they argued that the Msh2-dependent pathway was not functioning normally in GC cells. Together, these results would fit a simple unified model in which the expression of a GC/B cell-specific factor commandeers MMR and transforms this DNA repair pathway into a mutagenic pathway.An alternative explanation is that the MMR pathway is only mutagenic at sites mutated by AID, which would argue against a factor that disrupts global MMR function in GC B cells. To explore this issue, we examined the impact of Msh2-deficiency at a lacI transgene that is not mutated by AID in GC B cells. Our results indicate that Msh2 efficiently repairs lacI mutations in all cell populations examined, including GC B cells. Thus, MMR functions normally in GC ...

Elevated Incidence of Polyp Formation in APCMin/+Msh2−/− Mice Is Independent of Nitric Oxide-Induced DNA Mutations

“…We next examined the characteristics of mutations in the lacI gene in macrophages that were exposed to high amounts of nitric oxide. These mutations were compared to the mutation spectrum in WT and Msh2 −/− macrophages that were not treated with SNAP [36]. The background mutant frequency was increased ∼2-fold in SNAP treated Msh2 −/− macrophages (Figure 1B).…”

“…Msh2 −/− mice were crossed with Big Blue (BB + ) to generate BB + Msh2 +/− and BB + Msh2 −/− offspring. The mice that carry the shuttle vector (BB+) were identified by PCR as we have already described [36]. The iNOS −/− mice were obtained from Dr. J Gommerman (Toronto).…”

AIDing antibody diversity by error-prone mismatch repair

“…This seems to confirm that the MMR pathway is highly mutagenic only at AID-targeted loci since it is much less mutagenic at a lacI transgene that is not targeted by AID [14]. Additional studies of the protein complexes and signaling cascades that accompany AID activity and of the genetic and epigenetic peculiarities of the Ig genes could reveal how MMR mediated mutagenesis is largely restricted to the Ig genes.…”

“…However, subsequent studies revealed that many other genes were mutated in activated B cells some of which were repaired with high fidelity while others were also subjected to error-prone repair [14, 15] (see Saribasak and Gearhart this issue ). The recent report that there are ~1 million sites occupied by AID in activated mouse B cells is surprising considering its genotoxic potential [16].…”

“…As Illustrated in Figure 1, during normal DNA replication the resynthesis of the excised strand is carried out by high fidelity polymerases δ and ε, but at the V – and possibly S regions – Polη is recruited by mono-ubiquitylated PCNA [45, 46]. The length of this patch has been estimated to be 20-30 bp [14, 15, 47]. It is unclear what restricts the patch to this size since in biochemical studies much longer strands of DNA can be excised by EXO1 [48, 49].…”