A role for Msh6 but not Msh3 in somatic hypermutation and class switch recombination - PubMed (original) (raw)

A role for Msh6 but not Msh3 in somatic hypermutation and class switch recombination

Stella A Martomo et al. J Exp Med. 2004.

Abstract

Somatic hypermutation is initiated by activation-induced cytidine deaminase (AID), and occurs in several kilobases of DNA around rearranged immunoglobulin variable (V) genes and switch (S) sites before constant genes. AID deaminates cytosine to uracil, which can produce mutations of C:G nucleotide pairs, and the mismatch repair protein Msh2 participates in generating substitutions of downstream A:T pairs. Msh2 is always found as a heterodimer with either Msh3 or Msh6, so it is important to know which one is involved. Therefore, we sequenced V and S regions from Msh3- and Msh6-deficient mice and compared mutations to those from wild-type mice. Msh6-deficient mice had fewer substitutions of A and T bases in both regions and reduced heavy chain class switching, whereas Msh3-deficient mice had normal antibody responses. This establishes a role for the Msh2-Msh6 heterodimer in hypermutation and switch recombination. When the positions of mutation were mapped, several focused peaks were found in Msh6(-/-) clones, whereas mutations were dispersed in Msh3(-/-) and wild-type clones. The peaks occurred at either G or C in WGCW motifs (W = A or T), indicating that C was mutated on both DNA strands. This suggests that AID has limited entry points into V and S regions in vivo, and subsequent mutation requires Msh2-Msh6 and DNA polymerase.

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Figures

Figure 1.

Figure 1.

Diminished heavy chain class switching in Msh6 −/− but not Msh3 −/− B cells. (A) Flow cytometry analysis. Spleen cells were stimulated with LPS or LPS plus IL-4 to induce switching, and surface immunoglobulin was measured 3 d later. The percentage of B220+ cells that switched to IgG3 or IgG1 is shown in each box. (B) Serum antibody analysis. An ELISA was used to quantify anti-KLH antibodies from immunized mice. Black bars, C57BL/6; white bars, Msh6 −/−. Units are defined as the antibody titer that was normalized to the total amount of each isotype.

Figure 2.

Figure 2.

Fewer A:T substitutions in JH4 introns from Msh6 −/− clones. (A) The total number of clones analyzed is shown in the center of each circle. Segments represent the proportion of clones that contain the indicated number of mutations. (B) The sequence contains 26% A, 32% T, 28% G, and 14% C; values were corrected to represent a sequence with equal amounts of the four nucleotides. Mutations are shown from the nontranscribed strand. (C) Total mutations for each nucleotide are grouped.

Figure 3.

Figure 3.

Reduced A:T mutations in Sμ from Msh6 −/− clones. (A) The proportion of clones containing mutations is shown. (B) Values are corrected for nucleotide composition of the sequence, which contained 33% A, 23% T, 28% G, and 16% C. (C) Total mutations of each nucleotide are grouped.

Figure 4.

Figure 4.

Distribution of mutations in JH4 intron reveals peaks in Msh6 −/− clones. Nucleotides are numbered from the first base after the JH4 coding sequence, with 1 corresponding to 2,340 of Genbank/EMBL/DDBJ under accession no. J00440. Data were calculated as the number of mutations at a nucleotide position divided by the number of mutated clones. Bars below the abscissa depict WGCW motifs.

Figure 5.

Figure 5.

Location of mutations in Sμ shows targeting in Msh6 −/− clones. Nucleotides are numbered with 1 corresponding to 4,600 of Genbank/EMBL/DDBJ under accession no. J00440. Data represent the number of mutations at a position divided by the total number of clones. Bars below the abscissa represent WGCW motifs.

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