Altered spectra of hypermutation in antibodies from mice deficient for the DNA mismatch repair protein PMS2

Abstract

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...

The Targeting of Somatic Hypermutation Closely Resembles That of Meiotic Mutation

“…Several studies have found reduced mutation rates in mismatch repair-deficient mice (11,14,16) and relative enhancement of mutations at the AGC/GCT hot spots (16) or at G and C bases (13,15). Rada et al (16) inferred from this observation that the mutator has two components, one that is dependent on the mismatch repair protein MSH-2 and another that is MSH-2 independent.…”

Somatic hypermutation of immunoglobulin and non–immunoglobulin genes

“…There is considerable evidence that DNA MMR has an interplay with SHM (Cascalho et al 1998;Frey et al 1998;Jacobs et al 1998;Kim et al 1999;Kim & Storb 1998;Phung et al 1998;Rada et al 1998;Winter et al 1998). In our view, MMR is not involved in creating the mutations since in MMR-de¢cient mice the frequency of mutations and the mutation hot spots and cold spots are not signi¢-cantly altered (Kim et al 1999).…”

Nucleotide excision repair in an immunoglobulin variable gene is less efficient than in a housekeeping gene

“…This pathway does not directly contribute to any of the V gene diversification mechanisms discussed above, since V-D-J joining, hypermutation, and class switch recombination were normal in animals deficient for repair factors XPA (Winter et al, 1998), XPB (Kim et al, 1997), XPC (Shen et al, 1997), XPD (Wagner et al, 1996), XPF (Tian et al, 2004a), XPG (Tian et al, 2004b), CSA (Kim et al, 1997), and CSB (Jacobs et al, 1998), with the exception of ERCC1 (Schrader et al, 2004). However, it was used as an experimental tool because the nucleotide excision repair capacity in a specific genomic region may reflect unusual aspects of the locus.…”