The RAG1 N-terminal region regulates the efficiency and pathways of synapsis for V(D)J recombination.

Immunoglobulin and T cell receptor gene assembly depends on V(D)J recombination initiated by the RAG1-RAG2 recombinase. The RAG1 N-terminal region (NTR; aa 1-383) has been implicated in regulatory functions whose influence on V(D)J recombination and lymphocyte development in vivo is poorly understood. We generated mice in which RAG1 lacks ubiquitin ligase activity (P326G), the major site of autoubiquitination (K233R), or its first 215 residues (Δ215). While few abnormalities were detected in R1.K233R mice, R1.P326G mice exhibit multiple features indicative of reduced recombination efficiency, including an increased Igκ+:Igλ+ B cell ratio and decreased recombination of Igh, Igκ, Igλ, and Tcrb loci. Previous studies indicate that synapsis of recombining partners during Igh recombination occurs through two pathways: long-range scanning and short-range collision. We find that R1Δ215 mice exhibit reduced short-range Igh and Tcrb D-to-J recombination. Our findings indicate that the RAG1 NTR regulates V(D)J recombination and lymphocyte development by multiple pathways, including control of the balance between short- and long-range recombination.

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

DNA polymerase ι (Pol ι) is an attractive candidate for somatic hypermutation in antibody genes because of its low fidelity. To identify a role for Pol ι, we analyzed mutations in two strains of mice with deficiencies in the enzyme: 129 mice with negligible expression of truncated Pol ι, and knock-in mice that express full-length Pol ι that is catalytically inactive. Both strains had normal frequencies and spectra of mutations in the variable region, indicating that loss of Pol ι did not change overall mutagenesis. We next examined if Pol ι affected tandem mutations generated by another error-prone polymerase, Pol ζ. The frequency of contiguous mutations was analyzed using a novel computational model to determine if they occur during a single DNA transaction or during two independent events. Analyses of 2,000 mutations from both strains indicated that Pol ι-compromised mice lost the tandem signature, whereas C57BL/6 mice accumulated significant amounts of double mutations. The results support a model where Pol ι occasionally accesses the replication fork to generate a first mutation, and Pol ζ extends the mismatch with a second mutation.

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.

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.

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.

DNA polymerase ζ generates tandem mutations in immunoglobulin variable regions.

Low-fidelity DNA polymerases introduce nucleotide substitutions in immunoglobulin variable regions during somatic hypermutation. Although DNA polymerase (pol) η is the major low-fidelity polymerase, other DNA polymerases may also contribute. Existing data are contradictory as to whether pol ζ is involved. We reasoned that the presence of pol η may mask the contribution of pol ζ, and therefore we generated mice deficient for pol η and heterozygous for pol ζ. The frequency and spectra of hypermutation was unaltered between Polζ(+/-) Polη(-/-) and Polζ(+/+) Polη(-/-) clones. However, there was a decrease in tandem double-base substitutions in Polζ(+/-) Polη(-/-) cells compared with Polζ(+/+) Polη(-/-) cells, suggesting that pol ζ generates tandem mutations. Contiguous mutations are consistent with the biochemical property of pol ζ to extend a mismatch with a second mutation. The presence of this unique signature implies that pol ζ contributes to mutational synthesis in vivo. Additionally, data on tandem mutations from wild type, Polζ(+/-), Polζ(-/-), Ung(-/-), Msh2(-/-), Msh6(-/-), and Ung(-/-) Msh2(-/-) clones suggest that pol ζ may function in the MSH2-MSH6 pathway.

XRCC1 suppresses somatic hypermutation and promotes alternative nonhomologous end joining in Igh genes.

Activation-induced deaminase (AID) deaminates cytosine to uracil in immunoglobulin genes. Uracils in DNA can be recognized by uracil DNA glycosylase and abasic endonuclease to produce single-strand breaks. The breaks are repaired either faithfully by DNA base excision repair (BER) or mutagenically to produce somatic hypermutation (SHM) and class switch recombination (CSR). To unravel the interplay between repair and mutagenesis, we decreased the level of x-ray cross-complementing 1 (XRCC1), a scaffold protein involved in BER. Mice heterozygous for XRCC1 showed a significant increase in the frequencies of SHM in Igh variable regions in Peyer's patch cells, and of double-strand breaks in the switch regions during CSR. Although the frequency of CSR was normal in Xrcc1(+/-) splenic B cells, the length of microhomology at the switch junctions decreased, suggesting that XRCC1 also participates in alternative nonhomologous end joining. Furthermore, Xrcc1(+/-) B cells had reduced Igh/c-myc translocations during CSR, supporting a role for XRCC1 in microhomology-mediated joining. Our results imply that AID-induced single-strand breaks in Igh variable and switch regions become substrates simultaneously for BER and mutagenesis pathways.

Uracil residues dependent on the deaminase AID in immunoglobulin gene variable and switch regions.

Activation-induced deaminase (AID) initiates diversity of immunoglobulin genes through deamination of cytosine to uracil. Two opposing models have been proposed for the deamination of DNA or RNA by AID. Although most data support DNA deamination, there is no physical evidence of uracil residues in immunoglobulin genes. Here we demonstrate their presence by determining the sensitivity of DNA to digestion with uracil DNA glycosylase (UNG) and abasic endonuclease. Using several methods of detection, we identified uracil residues in the variable and switch regions. Uracil residues were generated within 24 h of B cell stimulation, were present on both DNA strands and were found to replace mainly cytosine bases. Our data provide direct evidence for the model that AID functions by deaminating cytosine residues in DNA.

Local sequence targeting in the AID/APOBEC family differentially impacts retroviral restriction and antibody diversification.

Nucleic acid cytidine deaminases of the activation-induced deaminase (AID)/APOBEC family are critical players in active and innate immune responses, playing roles as target-directed, purposeful mutators. AID specifically deaminates the host immunoglobulin (Ig) locus to evolve antibody specificity, whereas its close relative, APOBEC3G (A3G), lethally mutates the genomes of retroviral pathogens such as HIV. Understanding the basis for the target-specific action of these enzymes is essential, as mistargeting poses significant risks, potentially promoting oncogenesis (AID) or fostering drug resistance (A3G). AID prefers to deaminate cytosine in WRC (W = A/T, R = A/G) motifs, whereas A3G favors deamination of CCC motifs. This specificity is largely dictated by a single, divergent protein loop in the enzyme family that recognizes the DNA sequence. Through grafting of this substrate-recognition loop, we have created enzyme variants of A3G and AID with altered local targeting to directly evaluate the role of sequence specificity on immune function. We find that grafted loops placed in the A3G scaffold all produced efficient restriction of HIV but that foreign loops in the AID scaffold compromised hypermutation and class switch recombination. Local targeting, therefore, appears alterable for innate defense against retroviruses by A3G but important for adaptive antibody maturation catalyzed by AID. Notably, AID targeting within the Ig locus is proportionally correlated to its in vitro ability to target WRC sequences rather than non-WRC sequences. Although other mechanisms may also contribute, our results suggest that local sequence targeting by AID/APOBEC3 enzymes represents an elegant example of co-evolution of enzyme specificity with its target DNA sequence.

Hijacked DNA repair proteins and unchained DNA polymerases.

Somatic hypermutation of immunoglobulin (Ig) genes occurs at a frequency that is a million times greater than the mutation in other genes. Mutations occur in variable genes to increase antibody affinity, and in switch regions before constant genes to cause switching from IgM to IgG. Hypermutation is initiated in activated B cells when the activation-induced deaminase protein deaminates cytosine in DNA to uracil. Uracils can be processed by either a mutagenic pathway to produce mutations or a non-mutagenic pathway to remove mutations. In the mutagenic pathway, we first studied the role of mismatch repair proteins, MSH2, MSH3, MSH6, PMS2 and MLH1, since they would recognize mismatches. The MSH2-MSH6 heterodimer is involved in hypermutation by binding to U:G and other mismatches generated during repair synthesis, but the other proteins are not necessary. Second, we analysed the role of low-fidelity DNA polymerases eta, iota and theta in synthesizing mutations, and conclude that polymerase eta is the dominant participant by generating mutations at A:T base pairs. In the non-mutagenic pathway, we examined the role of the Cockayne syndrome B protein that interacts with other repair proteins. Mice deficient in this protein had normal hypermutation and class switch recombination, showing that it is not involved.

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.

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.

Uracil DNA glycosylase disruption blocks Ig gene conversion and induces transition mutations.

Ig gene conversion is most likely initiated by activation-induced cytidine deaminase-mediated cytosine deamination. If the resulting uracils need to be further processed by uracil DNA glycosylase (UNG), UNG inactivation should block gene conversion and induce transition mutations. In this study, we report that this is indeed the phenotype in the B cell line DT40. Ig gene conversion is almost completely extinguished in the UNG-deficient mutant and large numbers of transition mutations at C/G bases accumulate within the rearranged Ig L chain gene (IgL). The mutation rate of UNG-deficient cells is about seven times higher than that of pseudo V gene-deleted (psiV-) cells in which mutations arise presumably after uracil excision. In addition, UNG-deficient cells show relatively more mutations upstream and downstream of the VJ segment. This suggests that hypermutating B cells process activation-induced cytidine deaminase-induced uracils with approximately one-seventh of uracils giving rise to mutations depending on their position.

Basic cell culture conditions.

The DT40 cell line is derived from ALV (avian leukosis virus)-transformed bursal B cell of chicken. This cell line is suspension cell, and can be cultured in cell culture bottles and in culture plates (6-, 24- and 96-well etc.). Optimal culture temperature for the DT40 is (39 degrees C-) 41 degrees C, because chicken has higher body temperature than human and mouse. Here we summarize protocols for culture medium and condition of the DT40 cell line.

Targeted transfection of DT40 cells.

In order to understand the function of genes, transfection can be used as a method in which artificially prepared knockout and expression constructs are being introduced into cell lines. Since many genes are essential for embryonic development and a homozygous deletion results in non-viable embryos, gene disruption in a cell line by using transfected constructs might be an alternative choice. Electroporation is often used to stably transfect knockout and knockin vectors into DT40 cell line.

A follow-up study on the effect of Helicobacter pylori eradication on the severity of gastric histology.

Helicobacter pylori genetic diversity and geographic distribution affect the severity of gastric histology; while eradication heals gastritis, the improvement of atrophy and intestinal metaplasia (IM) is still controversial. We investigated whether H. pylori infection and genotypes (cagA-vacA) influence the histological changes and whether eradication resolves these changes. Twenty-one patients (11 duodenal ulcer, 2 gastric ulcer, 8 gastritis) received treatment. Biopsies for CLO, PCR, histology, and culture were collected before and at 1 and 12 months after treatment, and serum samples at 0, 1, 2, 6, and 12 months. H. pylori eradication was achieved in 71% of the patients. Histological scores for H. pylori densities were significantly higher in the antrum and incisura angularis. Scores for mononuclear cell and neutrophil infiltration were significantly higher in regions with a high H. pylori density and improved progressively after eradication. Eight patients with atrophy including five with IM showed no significant changes 12 months after eradication. The cagA gene, detected in 13 (62%), the vacA-sla gene, in 20 (95%), and the vacA-m1 gene, in 12 (57%) of 21 patients were significantly associated with duodenal ulcer. A gradual decline in antibody titer reached an average of 67% 12 months after eradication. H. pylori infection and the associated genotypes (cagA of Western type) affect the severity of the gastric histology (mild forms of atrophy and IM) and the disease outcome. Eradication of H. pylori resulted in healing of gastritis, but with no significant improvement in atrophy or IM.

Activation-induced cytidine deaminase initiates immunoglobulin gene conversion and hypermutation by a common intermediate.

Depending on the species and the lymphoid organ, activation-induced cytidine deaminase (AID) expression triggers diversification of the rearranged immunoglobulin (Ig) genes by pseudo V (psiV) gene- templated gene conversion or somatic hypermutation. To investigate how AID can alternatively induce recombination or hypermutation, psiV gene deletions were introduced into the rearranged light chain locus of the DT40 B-cell line. We show that the stepwise removal of the psiV donors not only reduces and eventually abolishes Ig gene conversion, but also activates AID-dependent Ig hypermutation. This strongly supports a model in which AID induces a common modification in the rearranged V(D)J segment, leading to a conversion tract in the presence of nearby donor sequences and to a point mutation in their absence.

Analysis of Helicobacter pylori genotypes and correlation with clinical outcome in Turkey.

The predominant Helicobacter pylori strains circulating among geographic locations differ in regard to genomic structure. The association of the cagA-positive, vacA s1 genotypes with peptic ulcer disease (PUD) and gastric cancer was reported in Western countries but not in East Asian countries. Strains from Western countries predominantly possessed cagA type 2a, vacA s1a or s1b/m1a, or vacA m2a genotypes, whereas strains from East Asia possessed cagA type 1a, vacA s1c/m1b, or vacA m2b genotypes. Whether the Turkish strains possessed such genotypes was investigated and correlated with the disease outcome. Seventy-three patients from Turkey were enrolled. H. pylori was detected in 65 (89%) patients (22 with gastritis, 33 with PUD, and 10 with gastric cancer) by any of the following tests: Campylobacter-like organism test, culture, or PCR. Among the H. pylori-positive patients, presence of the cagA gene (78%) was significantly associated with PUD (P < 0.00001), gastric cancer (P < 0.001), and vacA s1a genotypes (P < 0.0001). Multiple vacA genotypes were more prevalent in PUD and gastric cancer patients than in patients with gastritis. Restriction fragment length polymorphism analysis of the cagA gene revealed three different patterns with no significant association with clinical outcome. Turkish strains examined predominantly possessed cagA type 2a, vacA s1a/m1a, or vacA m2a genotypes, which were typical genotypes in strains from Western countries. This fact might be one of the reasons for the low prevalence of severe gastroduodenal diseases in Turkey compared to the East Asian countries.