Different mismatch repair deficiencies all have the same effects on somatic hypermutation: intact primary mechanism accompanied by secondary modifications

Abstract

Somatic hypermutation of Ig genes is probably dependent on transcription of the target gene via a mutator factor associated with the RNA polymerase (Storb, U., E.L. Klotz, J. Hackett, Jr., K. Kage, G. Bozek, and T.E. Martin. 1998. J. Exp. Med. 188:689-698). It is also probable that some form of DNA repair is involved in the mutation process. It was shown that the nucleotide excision repair proteins were not required, nor were mismatch repair (MMR) proteins. However, certain changes in mutation patterns and frequency of point mutations were observed in Msh2 (MutS homologue) and Pms2 (MutL homologue) MMR-deficient mice (for review see Kim, N., and U. Storb. 1998. J. Exp. Med. 187:1729-1733). These data were obtained from endogenous immunoglobulin (Ig) genes and were presumably influenced by selection of B cells whose Ig genes had undergone certain mutations. In this study, we have analyzed somatic hypermutation in two MutL types of MMR deficiencies, Pms2 and Mlh1. The mutation target was a nonselectable Ig-kappa gene with an artificial insert in the V region. We found that both Pms2- and Mlh1-deficient mice can somatically hypermutate the Ig test gene at approximately twofold reduced frequencies. Furthermore, highly mutated sequences are almost absent. Together with the finding of genome instability in the germinal center B cells, these observations support the conclusion, previously reached for Msh2 mice, that MMR-deficient B cells undergoing somatic hypermutation have a short life span. Pms2- and Mlh-1-deficient mice also resemble Msh2-deficient mice with respect to preferential targeting of G and C nucleotides. Thus, it appears that the different MMR proteins do not have unique functions with respect to somatic hypermutation. Several intrinsic characteristics of somatic hypermutation remain unaltered in the MMR-deficient mice: a preference for targeting A over T, a strand bias, mutational hot spots, and hypermutability of the artificial insert are all seen in the unselectable Ig gene. This implies that the MMR proteins are not required for and most likely are not involved in the primary step of introducing the mutations. Instead, they are recruited to repair certain somatic point mutations, presumably soon after these are created.

Figure 1

Analysis of VH11 gene mutations. A VH11 sequence is represented by each line. The locations of mutations are indicated by arrows with the specific nucleotide change written above each arrow. The solid line represents the sequenced region and the dotted line represents the unanalyzed sequence. All clones represented in this figure are of the IgG isotype. (A) VH11 clones from Mlh1^−/− mice. (B) VH11 clones from Pms2^−/− mice.

Figure 2

Map of the Vk167/PEPS transgene and EPS analysis. (A) Vk167/PEPS transgene and sequence of the EPS insert. The restriction sites within the EPS are indicated. (B) An example of PAGE from an EPS analysis. PCR products from nine clones were digested with EcoRV (E) or PvuII (P) and resolved in 18% acrylamide gel. Unmutated clones (12G, 2E, 2B, and 2D) show two large bands flanking the EPS region and ladders of small bands. In case of EcoRV digestion, the small bands are 20, 18, 16, 14, 12, and 10 bp (12- and 10-bp bands are invisible here). In case of PvuII digestion, the small bands are 19, 17, 15, 13, and 11 bp (11-bp bands are invisible). An asterisk indicates mutated clones. Clone 8G, for example, has a mutation in the fourth PvuII site (PD), so the 15- and 17-bp bands are gone and a 32-bp band has appeared.

Figure 2

Map of the Vk167/PEPS transgene and EPS analysis. (A) Vk167/PEPS transgene and sequence of the EPS insert. The restriction sites within the EPS are indicated. (B) An example of PAGE from an EPS analysis. PCR products from nine clones were digested with EcoRV (E) or PvuII (P) and resolved in 18% acrylamide gel. Unmutated clones (12G, 2E, 2B, and 2D) show two large bands flanking the EPS region and ladders of small bands. In case of EcoRV digestion, the small bands are 20, 18, 16, 14, 12, and 10 bp (12- and 10-bp bands are invisible here). In case of PvuII digestion, the small bands are 19, 17, 15, 13, and 11 bp (11-bp bands are invisible). An asterisk indicates mutated clones. Clone 8G, for example, has a mutation in the fourth PvuII site (PD), so the 15- and 17-bp bands are gone and a 32-bp band has appeared.

Figure 3

Analysis of Vk167/PEPS transgene mutations in splenic GC B lymphocytes. The original transgene sequences from NT 408 to 916 are shown in uppercase letters. Mutations are indicated in lowercase letters. The restriction sites within the EPS are indicated by a line over the sequence. E is an EcoRV and P is a PvuII site. A Δ denotes a deletion. (A) Mutations from Mlh1^−/− mice. (B) Mutations from Pms2^−/− mice.

Figure 3

Analysis of Vk167/PEPS transgene mutations in splenic GC B lymphocytes. The original transgene sequences from NT 408 to 916 are shown in uppercase letters. Mutations are indicated in lowercase letters. The restriction sites within the EPS are indicated by a line over the sequence. E is an EcoRV and P is a PvuII site. A Δ denotes a deletion. (A) Mutations from Mlh1^−/− mice. (B) Mutations from Pms2^−/− mice.

Figure 6

Microsatellite instability in Peyer's patch B lymphocytes of Mlh1^−/− mice. PNA^hi indicates the B220⁺PNA^hi cell population (GC B cells) and PNA^lo indicates the B220⁺PNA^lo cell population (non-GC B cells). Each lane contains the product of a single PCR reaction with about one cell equivalent of genomic DNA as template. Primers specific to D4Mit42 SSR locus are used in the PCR. Three out of nine separate reactions from the PNA^hi population show instability in CA repeats (indicated by an asterisk), as evidenced by the appearance of the shorter PCR product. All reactions from the PNA^lo population show the PCR products of the correct length (102 nucleotides).

Figure 4

Analysis of Vk167/PEPS transgene mutations in Peyer's patch GC B lymphocytes. (A) Mutations from wild-type mice. (B) Mutations from Mlh1^−/− mice.

Figure 4

Analysis of Vk167/PEPS transgene mutations in Peyer's patch GC B lymphocytes. (A) Mutations from wild-type mice. (B) Mutations from Mlh1^−/− mice.

Figure 5

Mutation accumulation in Peyer's patch B cells. Each bar represents the percentage of Vk167/PEPS clones with a given number of mutations. The distribution is calculated from the total number of clones analyzed as indicated in Table .

Figure 7

Mutation distribution within EcoRV or PvuII sites of the EPS sequence. Mutations from all six PvuII sites and all seven EcoRV sites are combined. Each bar represents the percentage of mutations at the specific location within the restriction recognition site out of total mutations. For example, the percentage of mutation at G nucleotide at position 3 of the PvuII site equals the number of mutations at G in all PvuII sites divided by the number of total mutations. Data from the analysis of splenic GC B cells and the analysis of Peyer's patch GC B cells were combined for this figure.