To determine whether DNA polymerase eta plays a role in the hypermutation of immunoglobulin variable genes, we examined the frequency and pattern of substitutions in variable VH6 genes from the peripheral blood lymphocytes of three patients with xeroderma pigmentosum variant disease, whose polymerase eta had genetic defects. The frequency of mutation was normal but the types of base changes were different: there was a decrease in mutations at A and T and a concomitant rise in mutations at G and C. We propose that more than one polymerase contributes to hypermutation and that if one is absent, others compensate. The data indicate that polymerase eta is involved in generating errors that occur predominantly at A and T and that another polymerase(s) may preferentially generate errors opposite G and C.

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...

SUMMARYThe third complementarity-determining region (CDR3) of immunoglobulin variable genes for the heavy chain (V H ) has been shown to be shorter in length in hypermutated antibodies than in nonhypermutated antibodies. To determine which components of CDR3 contribute to the shorter length, and if there is an effect of age on the length, we analysed 235 cDNA clones from human peripheral blood of V H 6 genes rearranged to immunoglobulin M (IgM) constant genes. There was similar use of diversity (D) and joining (J H ) gene segments between clones from young and old donors, and there was similar use of D segments among the mutated and non-mutated heavy chains. However, in the mutated heavy chains, there was increased use of shorter J H 4 segments and decreased use of longer J H 6 segments compared to the non-mutated proteins. The overall length of CDR3 did not change with age within the mutated and non-mutated categories, but was signi®cantly shorter by three amino acids in the mutated clones compared to the non-mutated clones. Analyses of the individual components that comprise CDR3 indicated that they were all shorter in the mutated clones. Thus, there were more nucleotides deleted from the ends of V H , D, and J H gene segments, and fewer P and N nucleotides added. The results suggest that B cells bearing immunoglobulin receptors with shorter CDR3s have been selected for binding to antigen. A smaller CDR3 may allow room in the antibody binding pocket for antigen to interact with CDRs 1 and 2 as well, so that as the VDJ gene undergoes hypermutation, substitutions in all three CDRs can further contribute to the binding energy.

The immunogenicity of recombinant protein or anti-viral DNA vaccines can be significantly improved by the addition of tandem copies of the complement fragment C3d. We sought to determine if the efficacy of a circumsporozoite protein (CSP)-based DNA vaccine delivered to mouse skin by gene gun was improved by using this strategy. Instead, we found that C3d suppressed the protective immunity against Plasmodium berghei malaria infection and deviated immunity, most notably by suppressing the induction of antibodies specific for the CSP C-terminal flanking sequence and by suppressing the induction of CSP-specific IL-4-producing spleen cells. We further showed that C3d bound to the C-terminal flanking sequence of the CSP, which may explain the immune deviation observed in CS/C3d chimeric antigen. We have thus identified C3d-mediated epitope masking and shifting of both the humoral and cellular immune responses as a potential novel escape mechanism, which plasmodia may use to divert the induction of protective immunity.

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.

The immunoglobulin loci are uniquely unstable regions of the genome which undergo as much mutation and selection in a matter of days as a species can undergo in generations of evolution. We have studied the mutational pattern and targeting of this unusual hypermutation process over the past 16 years. The pattern of somatic mutations in rearranged variable (V) genes differs from the pattern of meiotic mutations, indicating that a different mechanism generates hypermutation than generates spontaneous mutation. Hypermutations begin on the 5' end of rearranged V genes downstream of the transcription initiation site and continue through the V exon and into the 3'-flanking region before tapering off. Mutations are located randomly throughout the DNA sequence and exhibit strand bias. The targeting of mutations to the region in and around the rearranged V gene appears to require interactions between the promoter and downstream intronic DNA sequences. The same mechanism that initiates hypermutation around V genes may also produce double-strand breaks that catalyze homologous recombination between rearranged V genes on two chromosomal alleles. With this data we have built a model of hypermutation which predicts that V-region DNA is destabilized at the nuclear matrix during transcription and undergoes strand breaks.

The nucleotide sequence of one of the Escherichia coli tyrosine-transfer ribonucleic acids was determined in order to compare its sequence with that of yeast tyrosine-transfer ribonucleic acid. Forty-four positions of both transfer ribonucleic acids are occupied by the same nucleotides if they are arranged in the manner shown here. The information obtained suggests that the conformation of transfer ribonucleic acid molecules may be a greater contributing factor than a specific nucleotide sequence in the interaction of transfer ribonucleic acid with its corresponding aminoacyl-transfer ribonucleic acid synthetase.