Patricia J. Gearhart

Does DNA repair occur during somatic hypermutation

Activation-induced deaminase (AID) initiates a flood of DNA damage in the immunoglobulin loci, leading to abasic sites, single-strand breaks and mismatches. It is compelling that some proteins in the canonical base excision and mismatch repair pathways have been hijacked to increase mutagenesis during somatic hypermutation. Thus, the AID-induced mutagenic pathways involve a mix of DNA repair proteins and low fidelity DNA polymerases to create antibody diversity. In this review, we analyze the roles of base excision repair, mismatch repair, and mutagenesis during somatic hypermutation of rearranged variable genes. The emerging view is that faithful base excision repair occurs simultaneously with mutagenesis, whereas faithful mismatch repair is mostly absent.

Spt5 accumulation at variable genes distinguishes somatic hypermutation in germinal center B cells from ex vivo–activated cells

Maul et al. show that proteins associated with variable gene hypermutation, such as Spt5 and AID, are recruited to the initiating form of RNA polymerase II specifically in cells activated in germinal centers but not in culture.

Age-Associated B Cells Express a Diverse Repertoire of VH and Vκ Genes with Somatic Hypermutation

The origin and nature of age-associated B cells (ABCs) in mice are poorly understood. In this article, we show that their emergence required MHC class II and CD40/CD40L interactions. Young donor B cells were adoptively transferred into congenic recipients and allowed to remain for 1 mo in the absence of external Ag. B cells expressing the T-bet transcription factor, a marker for ABCs, were generated after multiple cell divisions from C57BL/6 donors but not from MHC class II– or CD40-deficient donors. Furthermore, old CD154 (CD40L)-deficient mice did not accrue ABCs, confirming that they arise primarily through T-dependent interactions. To determine what Igs ABCs express, we sequenced VH and Vκ rearranged genes from unimmunized 22-mo-old C57BL/6 mice and showed that they had a heterogeneous repertoire, which was comparable to that seen in old follicular and marginal zone B cell subsets. However, in contrast to the follicular and marginal zone cells, ABCs displayed significant somatic hypermutation. The mutation frequency was lower than found in germinal center cells after deliberate immunization, suggesting that ABCs have undergone mild stimulation from endogenous Ags over time. These observations show that quiescent ABCs are Ag-experienced cells that accumulate during T cell–dependent responses to diverse Ags during the life of an individual.

Antibody diversification caused by disrupted mismatch repair and promiscuous DNA polymerases.

The enzyme activation-induced deaminase (AID) targets the immunoglobulin loci in activated B cells and creates DNA mutations in the antigen-binding variable region and DNA breaks in the switch region through processes known, respectively, as somatic hypermutation and class switch recombination. AID deaminates cytosine to uracil in DNA to create a U:G mismatch. During somatic hypermutation, the MutSα complex binds to the mismatch, and the error-prone DNA polymerase η generates mutations at A and T bases. During class switch recombination, both MutSα and MutLα complexes bind to the mismatch, resulting in double-strand break formation and end-joining. This review is centered on the mechanisms of how the MMR pathway is commandeered by B cells to generate antibody diversity.

Defective Repair of Uracil Causes Telomere Defects in Mouse Hematopoietic Cells

Background: Telomeres may be susceptible to uracil misincorporation, which is removed by uracil DNA glycosylase (UNG). Results: UNG deficiency increases uracil in telomeres and causes alterations in multiple aspects of telomere maintenance in proliferating mouse hematopoietic cells. Conclusion: Accumulation of uracil interferes with telomere maintenance. Significance: UNG-initiated base excision repair is necessary for the preservation of telomere integrity. Uracil in the genome can result from misincorporation of dUTP instead of dTTP during DNA synthesis, and is primarily removed by uracil DNA glycosylase (UNG) during base excision repair. Telomeres contain long arrays of TTAGGG repeats and may be susceptible to uracil misincorporation. Using model telomeric DNA substrates, we showed that the position and number of uracil substitutions of thymine in telomeric DNA decreased recognition by the telomere single-strand binding protein, POT1. In primary mouse hematopoietic cells, uracil was detectable at telomeres, and UNG deficiency further increased uracil loads and led to abnormal telomere lengthening. In UNG-deficient cells, the frequencies of sister chromatid exchange and fragility in telomeres also significantly increased in the absence of telomerase. Thus, accumulation of uracil and/or UNG deficiency interferes with telomere maintenance, thereby underscoring the necessity of UNG-initiated base excision repair for the preservation of telomere integrity.

ATM deficiency promotes development of murine B-cell lymphomas that resemble diffuse large B-cell lymphoma in humans

The serine-threonine kinase ataxia-telangiectasia mutated (ATM) plays a central role in maintaining genomic integrity. In mice, ATM deficiency is exclusively associated with T-cell lymphoma development, whereas B-cell tumors predominate in human ataxia-telangiectasia patients. We demonstrate in this study that when T cells are removed as targets for lymphomagenesis and as mediators of immune surveillance, ATM-deficient mice exclusively develop early-onset immunoglobulin M(+) B-cell lymphomas that do not transplant to immunocompetent mice and that histologically and genetically resemble the activated B cell-like (ABC) subset of human diffuse large B-cell lymphoma (DLBCL). These B-cell lymphomas show considerable chromosomal instability and a recurrent genomic amplification of a 4.48-Mb region on chromosome 18 that contains Malt1 and is orthologous to a region similarly amplified in human ABC DLBCL. Of importance, amplification of Malt1 in these lymphomas correlates with their dependence on nuclear factor (NF)-κB, MALT1, and B-cell receptor (BCR) signaling for survival, paralleling human ABC DLBCL. Further, like some human ABC DLBCLs, these mouse B-cell lymphomas also exhibit constitutive BCR-dependent NF-κB activation. This study reveals that ATM protects against development of B-cell lymphomas that model human ABC DLBCL and identifies a potential role for T cells in preventing the emergence of these tumors.

Refining the Neuberger model: Uracil processing by activated B cells.

During the immune response, B cells undergo a programed mutagenic cascade to promote increased affinity and expanded antibody function. The two processes, somatic hypermutation (SHM) and class switch recombination (CSR), are initiated by the protein activation‐induced deaminase (AID), which converts cytosine to uracil in the immunoglobulin loci. The presence of uracil in DNA promotes DNA mutagenesis though a subset of DNA repair proteins. Two distinct mechanisms have been proposed to control uracil processing. The first is through base removal by uracil DNA glycosylase (UNG), and the second is through detection by the mismatch repair (MMR) complex MSH2/6. In a study published in this issue of European Journal of Immunology, Dingler et al. [Eur. J. Immunol. 2014. 44: 1925‐1935] examine uracil processing in B cells in the absence of UNG and SMUG1 glycosylases. Similar to UNG, SMUG1 is an uracil glycosylase which can remove the uracil base. While Smug1−/− mice show no clear deficiency in SHM or CSR, Ung−/−Smug1−/− mice display exacerbated phenotypes, suggesting a back‐up role for SMUG1 in antibody diversity. This new information expands the model of uracil processing in B cells and raises several interesting questions about the dynamic relationship between base excision repair and MMR.

DNA polymerase ι functions in the generation of tandem mutations during somatic hypermutation of antibody genes

Gearhart and collaborators address the long-standing question of the roles of error-prone DNA polymerases in somatic hypermutation of antibody genes.

ATAD5 Deficiency Decreases B Cell Division and Igh Recombination

Mammalian ATPase family AAA domain–containing protein 5 (ATAD5) and its yeast homolog enhanced level of genomic instability 1 are responsible for unloading proliferating cell nuclear antigen from newly synthesized DNA. Prior work in HeLa and yeast cells showed that a decrease in ATAD5 protein levels resulted in accumulation of chromatin-bound proliferating cell nuclear antigen, slowed cell division, and increased genomic instability. In this study, B cells from heterozygous (Atad5+/m) mice were used to examine the effects of decreased cell proliferation on Ab diversity. ATAD5 haploinsufficiency did not change the frequency or spectrum of somatic hypermutation in Ab genes, indicating that DNA repair and error-prone DNA polymerase η usage were unaffected. However, immunized Atad5+/m mice had decreased serum IgG1 Abs, demonstrating a functional effect on class switch recombination. The mechanism of this altered immune response was then examined following ex vivo stimulation of splenic B cells, where Atad5+/m cells accumulated in the S phase of the cell cycle and had reduced proliferation compared with wild-type cells. These haploinsufficient cells underwent a significant decline in activation-induced deaminase expression, resulting in decreased switch region DNA double-strand breaks and interchromosomal translocations in the Igh locus. Class switch recombination to several isotypes was also reduced in Atad5+/m cells, although the types of end-joining pathways were not affected. These results describe a defect in DNA replication that affects Igh recombination via reduced cell division.

Topoisomerase I deficiency causes RNA polymerase II accumulation and increases AID abundance in immunoglobulin variable genes.

Activation-induced deaminase (AID) is a DNA cytosine deaminase that diversifies immunoglobulin genes in B cells. Recent work has shown that RNA polymerase II (Pol II) accumulation correlates with AID recruitment. However, a direct link between Pol II and AID abundance has not been tested. We used the DT40 B-cell line to manipulate levels of Pol II by decreasing topoisomerase I (Top1), which relaxes DNA supercoiling in front of the transcription complex. Top1 was decreased by stable transfection of a short hairpin RNA against Top1, which produced an accumulation of Pol II in transcribed genes, compared to cells transfected with sh-control RNA. The increased Pol II density enhanced AID recruitment to variable genes in the λ light chain locus, and resulted in higher levels of somatic hypermutation and gene conversion. It has been proposed by another lab that AID itself might directly suppress Top1 to increase somatic hypermutation. However, we found that in both AID(+/+) and AID(-/-) B cells from DT40 and mice, Top1 protein levels were identical, indicating that the presence or absence of AID did not decrease Top1 expression. Rather, our results suggest that the mechanism for increased diversity when Top1 is reduced is that Pol II accumulates and recruits AID to variable genes.