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 VκOx1 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.

Mutations are introduced into rearranged Ig variable genes at a frequency of 10 ؊2 mutations per base pair by an unknown mechanism. Assuming that DNA repair pathways generate or remove mutations, the frequency and pattern of mutation will be different in variable genes from mice defective in repair. Therefore, hypermutation was studied in mice deficient for either the DNA nucleotide excision repair gene Xpa or the mismatch repair gene Pms2. High levels of mutation were found in variable genes from XPA-deficient and PMS2-deficient mice, indicating that neither nucleotide excision repair nor mismatch repair pathways generate hypermutation. However, variable genes from PMS2-deficient mice had significantly more adjacent base substitutions than genes from wild-type or XPA-deficient mice. By using a biochemical assay, we confirmed that tandem mispairs were repaired by wild-type cells but not by Pms2 ؊/؊ human or murine cells. The data indicate that tandem substitutions are produced by the hypermutation mechanism and then processed by a PMS2-dependent pathway.During somatic hypermutation of Ig variable (V) genes in B lymphocytes, a large number of nucleotide substitutions are introduced into a small area of the genome for the purpose of generating variant antibodies with high affinity for cognate antigens. Substitutions are distributed at a frequency of 10 Ϫ2 ͞bp over a 2-kilobase region of DNA that includes the rearranged V gene and noncoding flanking sequences (1-4). Transgenic mice with modified Ig genes have shown that both promoter and enhancer transcription elements are required for hypermutation, perhaps by making the region more accessible to proteins causing mutation (5-8). More than 90% of the mutations are base substitutions, and the rest are deletions and insertions (4, 9). An analysis of the substitutions shows that transitions occur more frequently than transversions, and there are fewer mutations of T than of the other three nucleotides (10). Thus, the substitutions are somewhat asymmetric on each of the two strands (11), but it is not known whether they are preferentially introduced into one strand and͞or preferentially removed from one strand. The presence of multiple mutations implies that DNA repair pathways are involved in generating or removing them. We therefore studied the role of nucleotide excision repair and mismatch repair on the frequency and pattern of hypermutation.A possible role for nucleotide excision repair in somatic hypermutation of V genes has been proposed. Nucleotide excision repair is especially efficient in actively transcribed genes (reviewed in ref. 12), and the generation of base substitutions by the hypermutation process has been broadly correlated with the extent of transcription (5). Furthermore, mutagenesis and transcription are linked in yeast (13). DNA damage in the V region, or transcriptional stalling at pause sites, might be envisaged to initiate nucleotide excision repair events with an occasional error occurring during gap filling (6). Mice lacking the xer...

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