Increased hypermutation at G and C nucleotides in immunoglobulin variable genes from mice deficient in the MSH2 mismatch repair protein

Abstract

Rearranged immunoglobulin variable genes are extensively mutated after stimulation of B lymphocytes by antigen. Mutations are likely generated by an error-prone DNA polymerase, and the mismatch repair pathway may process the mispairs. To examine the role of the MSH2 mismatch repair protein in hypermutation, Msh2-/- mice were immunized with oxazolone, and B cells were analyzed for mutation in their VkappaOx1 light chain genes. The frequency of mutation in the repair-deficient mice was similar to that in Msh2+/+ mice, showing that MSH2-dependent mismatch repair does not cause hypermutation. However, there was a striking bias for mutations to occur at germline G and C nucleotides. The results suggest that the hypermutation pathway frequently mutates G.C pairs, and a MSH2-dependent pathway preferentially corrects mismatches at G and C.

Figure 1

Detection of a neo insert in the Msh2 gene in B220⁺PNA⁺ splenic B cells from Msh2 ^−/− mice. PCR products were electrophoresed through an agarose gel and stained with ethidium bromide. Lane 1, 100-bp ladder; lanes 2–5, amplification of DNA with primers as noted.

Figure 2

Location of mutations in the V_κOx1 gene from Msh2 ^+/+ C57BL/6 and Msh2 ^−/− mice. The coding region of the V_κOx1 gene segment is shown; nucleotide and codon numbering is according to Kabat et al. (19). The 276-bp coding region contained 90% of all mutations occurring in the entire 466-bp sequence. Msh2 ^+/+ C57BL/6 substitutions (18) are displayed above the coding sequence and Msh2 ^−/− substitutions are shown below. Codons 34 (nt 97–99) and 36 (nt 103–105) are underlined.

Figure 3

Bias for mutations at G·C pairs in Msh2 ^−/− clones. Frequencies of mutation of germline nucleotide pairs to any other base were calculated after correction for base composition. P values for whether the mutation frequency in A·T pairs is equal to the mutation frequency in G·C pairs are shown.