Somatic hypermutation of immunoglobulin genes in Rad18 knockout mice

Somatic hypermutation (SHM) of immunoglobulin (Ig) genes is triggered by the activity of activation-induced cytidine deaminase (AID). AID induces DNA lesions in variable regions of Ig genes, and error-prone DNA repair mechanisms initiated in response to these lesions introduce the mutations that characterize SHM. Error-prone DNA repair in SHM is proposed to be mediated by low-fidelity DNA polymerases such as those that mediate trans-lesion synthesis (TLS); however, the mechanism by which these enzymes are recruited to AID-induced lesions remains unclear. Proliferating cell nuclear antigen (PCNA), the sliding clamp for multiple DNA polymerases, undergoes Rad6/Rad18-dependent ubiquitination in response to DNA damage. Ubiquitinated PCNA promotes the replacement of the replicative DNA polymerase stalled at the site of a DNA lesion with a TLS polymerase. To examine the potential role of Rad18-dependent PCNA ubiquitination in SHM, we analyzed Ig gene mutations in Rad18 knockout (KO) mice immunized with T cell-dependent antigens. We found that SHM in Rad18 KO mice was similar to wild-type mice, suggesting that Rad18 is dispensable for SHM. However, residual levels of ubiquitinated PCNA were observed in Rad18 KO cells, indicating that Rad18-independent PCNA ubiquitination might play a role in SHM.     

AID-targeting and hypermutation of non-immunoglobulin genes does not correlate with proximity to immunoglobulin genes in germinal center B cells

Upon activation, B cells divide, form a germinal center, and express the activation induced deaminase (AID), an enzyme that triggers somatic hypermutation of the variable regions of immunoglobulin (Ig) loci. Recent evidence indicates that at least 25% of expressed genes in germinal center B cells are mutated or deaminated by AID. One of the most deaminated genes, c-Myc, frequently appears as a translocation partner with the Ig heavy chain gene (Igh) in mouse plasmacytomas and human Burkitt's lymphomas. This indicates that the two genes or their double-strand break ends come into close proximity at a biologically relevant frequency. However, the proximity of c-Myc and Igh has never been measured in germinal center B cells, where many such translocations are thought to occur. We hypothesized that in germinal center B cells, not only is c-Myc near Igh, but other mutating non-Ig genes are deaminated by AID because they are near Ig genes, the primary targets of AID. We tested this "collateral damage" model using 3D-fluorescence in situ hybridization (3D-FISH) to measure the distance from non-Ig genes to Ig genes in germinal center B cells. We also made mice transgenic for human MYC and measured expression and mutation of the transgenes. We found that there is no correlation between proximity to Ig genes and levels of AID targeting or gene mutation, and that c-Myc was not closer to Igh than were other non-Ig genes. In addition, the human MYC transgenes did not accumulate mutations and were not deaminated by AID. We conclude that proximity to Ig loci is unlikely to be a major determinant of AID targeting or mutation of non-Ig genes, and that the MYC transgenes are either missing important regulatory elements that allow mutation or are unable to mutate because their new nuclear position is not conducive to AID deamination.     

DNA polymerases and somatic hypermutation of immunoglobulin genes

Somatic hypermutation of immunoglobulin variable genes, which increases antibody diversity, is initiated by the activation-induced cytosine deaminase (AID) protein. The current DNA-deamination model posits that AID deaminates cytosine to uracil in DNA, and that mutations are generated by DNA polymerases during replication or repair of the uracil residue. Mutations could arise as follows: by DNA replicating past the uracil; by removing the uracil with a uracil glycosylase and replicating past the resulting abasic site with a low-fidelity polymerase; or by repairing the uracil and synthesizing a DNA-repair patch downstream using a low-fidelity polymerase. In this review, we summarize the biochemical properties of specialized DNA polymerases in mammalian cells and discuss their participation in the mechanisms of hypermutation. Many recent studies have examined mice deficient in the genes that encode various DNA polymerases, and have shown that DNA polymerase H (POLH) contributes to hypermutation, whereas POLI, POLK and several other enzymes do not have major roles. The low-fidelity enzyme POLQ has been proposed as another candidate polymerase because it can efficiently bypass abasic sites and recent evidence indicates that it might participate in hypermutation.     

Somatic hypermutation of immunoglobulin genes: lessons from proliferating cell nuclear antigenK164R mutant mice

Proliferating cell nuclear antigen (PCNA) encircles DNA as a ring-shaped homotrimer and, by tethering DNA polymerases to their template, PCNA serves as a critical replication factor. In contrast to high-fidelity DNA polymerases, the activation of low-fidelity translesion synthesis (TLS) DNA polymerases seems to require damage-inducible monoubiquitylation (Ub) of PCNA at lysine residue 164 (PCNA-Ub). TLS polymerases can tolerate DNA damage, i.e. they can replicate across DNA lesions. The lack of proofreading activity, however, renders TLS highly mutagenic. The advantage is that B cells use mutagenic TLS to introduce somatic mutations in immunoglobulin (Ig) genes to generate high-affinity antibodies. Given the critical role of PCNA-Ub in activating TLS and the role of TLS in establishing somatic mutations in immunoglobulin genes, we analysed the mutation spectrum of somatically mutated immunoglobulin genes in B cells from PCNA^K164R knock-in mice. A 10-fold reduction in A/T mutations is associated with a compensatory increase in G/C mutations—a phenotype similar to Polη and mismatch repair-deficient B cells. Mismatch recognition, PCNA-Ub and Polη probably act within one pathway to establish the majority of mutations at template A/T. Equally relevant, the G/C mutator(s) seems largely independent of PCNA^K164 modification.     

Somatic mutation of immunoglobulin light-chain variable-region genes

A single germline immunoglobulin kappa-variable-region gene, V_κ167, is rearranged and expressed in two myelomas, MOPC167 and MOPC511. Only this single germline gene displays close homology to the expressed genes. Neither of the rearranged, functional genes, however, has a nucleotide sequence that is identical to the germline V_κ167 gene. Both active genes display several single-base-pair mutations with respect to the germline sequence. The nucleotide sequence data predict the alteration of a restriction-enzyme-recognition site within the V_κ167 gene between germline cells and cells producing the MOPC167 light-chain protein. Based on this restriction-site alteration, Southern blot analysis proves unambiguously that no gene present in the germline BALB/c mouse genome contains the exact V_κ167 nucleotide sequence found in cells committed to MOPC167 antibody production. Instead, the alterations found in the expressed MOPC167 and MOPC511 V-region genes have apparently arisen by a process of somatic mutation during cellular differentiation. Since nucleotide alterations are found in framework and hypervariable portions of the variable region, the mechanism of somatic mutation is not limited to hypervariable sequences. In addition, Southern blot hybridization indicates that the observed mutations did not arise by recombinational events, but are single-base-pair substitutions. Based on the distribution of mutations that have been found in expressed immunoglobulin variable-region genes, a model that links the introduction of somatic mutations to DNA replication during the V-J joining event is proposed.     

On the molecular mechanism of somatic hypermutation of rearranged immunoglobulin genes

Somatic hypermutation (SHM) diversifies the genes that encode immunoglobulin variable regions in antigen-activated germinal centre B lymphocytes. Available evidence strongly suggests that DNA deamination potentiates phase I SHM and subsequently triggers phase II SHM. A concise review of this evidence is followed by a detailed critique of two possible models which suggest that polymerase-eta potentiates phase II SHM via either its DNA-dependent or its RNA-dependent DNA synthetic activity. Quantitative analysis, in the context of extant data that define the features of SHM, favours the RNA-dependent mechanism.     

Epstein-Barr virus and the somatic hypermutation of immunoglobulin genes in Burkitt's lymphoma cells

It has been suggested that Epstein-Barr virus (EBV) might suppress antibody maturation either by facilitating bypass of the germinal center reaction or by inhibiting hypermutation directly. However, by infecting the Burkitt's lymphoma (BL) cell line Ramos, which hypermutates constitutively and can be considered a transformed analogue of a germinal center B cell, with EBV as well as by transfecting it with selected EBV latency genes, we demonstrate that expression of EBV gene products does not lead to an inhibition of hypermutation. Moreover, we have identified two natural EBV-positive BL cell lines (ELI-BL and BL16) that hypermutate constitutively. Thus, contrary to expectations, EBV gene products do not appear to affect somatic hypermutation.     

Reevaluation of the role of DNA polymerase theta in somatic hypermutation of immunoglobulin genes

DNA polymerase theta has been implicated in the process of somatic hypermutation in immunoglobulin variable genes based on several reports of alterations in the frequency and spectra of mutations from Polq(-/-) mice. However, these studies have contrasting results on mutation frequencies and the types of nucleotide substitutions, which question the role of polymerase theta in hypermutation. DNA polymerase eta has a dominant effect on mutation and may substitute in the absence of polymerase theta to affect the pattern. Therefore, we have examined mutation in mice deficient for both polymerases theta and eta. The mutation frequencies in rearranged variable genes from Peyer's patches were similar in wild type, Polq(-/-), Polh(-/-), and Polq(-/-)Polh(-/-) mice. The types of substitutions were also similar between wild type and Polq(-/-) clones, and between Polh(-/-) and Polq(-/-)Polh(-/-) clones. Furthermore, there was no difference in heavy chain class switching in splenic B cells from the four groups of mice. These results indicate that polymerase theta does not play a significant role in the generation of somatic mutation in immunoglobulin genes.     

Might gene conversion be the mechanism of somatic hypermutation of mammalian immunoglobulin genes?

Gene conversion has played a major role in molding eukaryotic genomes, and this same mechanism mediates targeted sequence diversification of a variety of genes in response to developmental or environmental stimuli. Here I review data indicating that gene conversion may also be the molecular mechanism of somatic hypermutation at the mammalian immunoglobulin loci.     

Mitogen-driven B cell proliferation and differentiation are not accompanied by hypermutation of immunoglobulin variable region genes

Hybridomas were constructed from splenic B cells after mitogen stimulation in vitro with lipopolysaccharide and dextran sulfate for 9 to 11 days. Extensive proliferation and differentiation (secretion of IgG isotypes) was evident in these cultures before fusion. Hybridomas that express a VH gene segment whose germ-line sequence is known were isolated, and the nucleotide sequences of these expressed VH genes were determined. A total of 3775 VH nucleotides was analyzed in this way, and only one difference from the germ-line VH sequence was observed. The rate of V gene somatic mutation that has been estimated to occur during antigen-driven immune responses in vivo is 10(-3)/base pair/cell division. Given an estimated value for the number of cell divisions that occurred before hybridoma formation, at least 15 changes from the germ-line VH sequence should have been observed if mutation had been occurring at the in vivo rate during the culture period. Therefore, the data suggest that mitogen-driven B cell proliferation and differentiation are not sufficient to induce the hypermutation of Ig V region genes.     

Somatic hypermutation of immunoglobulin kappa may depend on sequences 3'' of C kappa and occurs on passenger transgenes.

We have compared the pattern of somatic mutation in different immunoglobulin kappa transgenes and suggest that an element(s) located between 1 kb and 9 kb 3' of C kappa is necessary for somatic hypermutation of the antibody V gene. The sequences of transgenic and endogenous Ig V regions were determined in antigen-specific B cell hybridomas specific for 2-phenyloxazolone from independent lines of hyperimmunized transgenic mice. We analysed somatic mutation of the transgene both in hybridomas in which the transgenic kappa chain contributes to the antigen combining site as well as in hybridomas in which the transgene is a passenger with the expressed antibody being composed of endogenously-encoded heavy and light chains. In both cases, nucleotide changes in the transgene are correctly targeted to the V region and are absent from the C region. They accumulate at a similar rate to that in the endogenous Ig genes within the same cell and we find that, irrespective of whether or not the transgene kappa is directly selected by antigen, somatic mutation occurs at a similar rate and involves only single base substitutions. Furthermore, the pattern of mutations in passenger transgenes gives information about the intrinsic sequence specificities of the somatic hypermutation mechanism.     

Somatic Hypermutation: Another piece in the hypermutation puzzle

Studies with transgenic mice are beginning to define the minimal requirements for the somatic hypermutation of immunoglobulin genes that is critical in the production of high-affinity antibodies.     

Brca1 in immunoglobulin gene conversion and somatic hypermutation

Defects in Brca1 confer susceptibility to breast cancer and genomic instability indicative of aberrant repair of DNA breaks. Brca1 was previously implicated in the homologous recombination pathway via effects on the assembly of recombinase Rad51. Activation-induced cytidine deaminase (AID) deaminates C to U in B lymphocyte immunoglobulin (Ig) DNA to initiate programmed DNA breaks. Subsequent uracil-glycosylase mediated U removal, and perhaps further processing, leads to four known classes of mutation: Ig class switch recombination that results in a region-specific genomic deletion, Ig somatic hypermutation that introduces point mutations in Ig V-regions, Ig gene conversion in vertebrates that possess Ig pseudo-V genes, and translocations common to B cell lymphomas. We tested the involvement of Brca1 in AID-dependent Ig diversification in chicken DT40 cells. The DT40 cell line diversifies IgVlambda mainly by gene conversion, and less so by point mutation. Brca1-deficiency caused a shift in Vlambda diversification, significantly reducing the proportion of gene conversions relative to point mutations. Thus, Brca1 regulates AID-dependent DNA lesion repair. Interestingly, while Brca1 is required to recruit ubiquitinated FancD2 to DNA damage, the phenotype of Brca1-deficient DT40 differs from the one of FancD2-deficient DT40, in which both gene conversion and non-templated mutations are impaired.     

A role for PCNA ubiquitination in immunoglobulin hypermutation

Proliferating cell nuclear antigen (PCNA) is a DNA polymerase cofactor and regulator of replication-linked functions. Upon DNA damage, yeast and vertebrate PCNA is modified at the conserved lysine K164 by ubiquitin, which mediates error-prone replication across lesions via translesion polymerases. We investigated the role of PCNA ubiquitination in variants of the DT40 B cell line that are mutant in K164 of PCNA or in Rad18, which is involved in PCNA ubiquitination. Remarkably, the PCNA^K164R mutation not only renders cells sensitive to DNA-damaging agents, but also strongly reduces activation induced deaminase-dependent single-nucleotide substitutions in the immunoglobulin light-chain locus. This is the first evidence, to our knowledge, that vertebrates exploit the PCNA-ubiquitin pathway for immunoglobulin hypermutation, most likely through the recruitment of error-prone DNA polymerases.