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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The hypermutation cascade in Ig V genes can be initiated by deamination of cytosine in DNA to uracil by activation-induced cytosine deaminase and its removal by uracil-DNA glycosylase. To determine whether damage to guanine also contributes to hypermutation, we examined the glycosylase that removes oxidized guanine from DNA, 8-hydroxyguanine-DNA glycosylase (OGG1). OGG1 has been reported to be overexpressed in human B cells from germinal centers, where mutation occurs, and could be involved in initiating Ab diversity by removing modified guanines. In this study, mice deficient in Ogg1 were immunized, and V genes from the H and κ L chain loci were sequenced. Both the frequency of mutation and the spectra of nucleotide substitutions were similar in ogg1−/− and Ogg1+/+ clones. More importantly, there was no significant increase in G:C to T:A transversions in the ogg1−/− clones, which would be expected if 8-hydroxyguanine remained in the DNA. Furthermore, Ogg1 was not up-regulated in murine B cells from germinal centers. These findings show that hypermutation is unaffected in the absence of Ogg1 activity and indicate that 8-hydroxyguanine lesions most likely do not cause V gene mutations.

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Glycoprotein (GP)IIb/IIIa, an integrin complex found on the surface of platelets, is a receptor for fibrinogen and other ligands, and is involved in platelet aggregation. Because GPIIb is specifically expressed in megakaryocytes, we have studied the 52-flanking region of the rat (r) GPIIb gene as a model of a megakaryocyte-specific gene. The studies presented here used a rat marrow expression system, which allows the study of primary cells undergoing terminal differentiation into megakaryocytes. The determination of megakaryocyte-specific expression of DNA constructs was possible by immunomagnetically separating megakaryocytes from total bone marrow cells. Transient expression constructs, containing varying lengths of the 52-flanking region from -39 to -912 bp, localized a regulatory element between -460 and -439 bp upstream of the transcriptional start site. This region contains a GATA consensus binding element between -457 and -454 (GATA454). Further constructs demonstrated that this GATA binding element was indeed essential for expression. A 25-bp substitution, covering the region -450 to -426 immediately downstream of the GATA454, demonstrated that this region was essential for full expression, which suggests that this region may interact with the GATA454 site in promoting high-level lineage-specific expression. To define regulatory elements between the GATA454 and the transcriptional start site further, we tested additional constructs derived from the original -912 construct; each of which contained the GATA454 but had different 50-bp deletions from -450 to the start site. Virtually all of these constructs continued to show high-level tissue-specific expression. The deleted -150 to -101 construct had twice the level of expression of the full-length wild-type construct; therefore, this region may contain a negative regulatory element. Comparison of our data with expression studies performed with the 52-region of the human GPIIb gene using HEL cells, a cell line with some megakaryocytic properties, demonstrates significant differences, which may reflect our use of primary rate bone marrow cells. In particular, our study points to the importance of the GATA454 for high levels of GPIIb expression in developing megakaryocytes.

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During somatic hypermutation of Ig V genes, mismatched nucleotide substitutions become candidates for removal by the DNA mismatch repair pathway. Previous studies have shown that V genes from mice deficient for the MSH2 and PMS2 mismatch repair proteins have frequencies of mutation that are comparable with those from wild-type (wt) mice; however, the pattern of mutation is altered. Because the absence of MSH2 and PMS2 produced different mutational spectra, we examined the role of another protein involved in mismatch repair, MLH1, on the frequency and pattern of hypermutation. MLH1-deficient mice were immunized with oxazolone Ag, and splenic B cells were analyzed for mutations in their VκOx1 light chain genes. Although the frequency of mutation in MLH1-deficient mice was twofold lower than in wt mice, the pattern of mutation in Mlh1−/− clones was similar to wt clones. These findings suggest that the MLH1 protein has no direct effect on the mutational spectrum.