DNA-editing Enzyme Research Articles

Viral Double-Stranded RNA Triggers Ig Class Switching by Activating Upper Respiratory Mucosa B Cells through an Innate TLR3 Pathway Involving BAFF

Class switch DNA recombination (CSR) from IgM to IgG and IgA is crucial for antiviral immunity. Follicular B cells undergo CSR upon engagement of CD40 by CD40 ligand on CD4+ T cells. This T cell-dependent pathway requires 5-7 days, which is too much of a delay to block quickly replicating pathogens. To compensate for this limitation, extrafollicular B cells rapidly undergo CSR through a T cell-independent pathway that involves innate Ag receptors of the TLR family. We found that a subset of upper respiratory mucosa B cells expressed TLR3 and responded to viral dsRNA, a cognate TLR3 ligand. In the presence of dsRNA, mucosal B cells activated NF-kappaB, a transcription factor critical for CSR. Activation of NF-kappaB required TRIF (Toll/IL-1R domain-containing protein inducing IFN-beta), a canonical TLR3 adapter protein, and caused germline transcription of downstream CH genes as well as expression of AID (activation-induced cytidine deaminase), a DNA-editing enzyme essential for CSR. Subsequent IgG and IgA production was enhanced by BAFF (B cell-activating factor of the TNF family), an innate mediator released by TLR3-expressing mucosal dendritic cells. Indeed, these innate immune cells triggered IgG and IgA responses upon exposure to dsRNA. By showing active TLR3 signaling and ongoing CSR in upper respiratory mucosa B cells from patients with CD40 signaling defects, our findings indicate that viral dsRNA may initiate frontline IgG and IgA responses through an innate TLR3-dependent pathway involving BAFF.

Quantitative Assessment of DNA Editing Enzymes in B-Cell Lymphomas.

Somatic hypermutation of immunoglobulin genes (SHM) is a physiological process that helps generate high affinity antibodies in germinal center B cells, and error-prone DNA polymerases are key enzymes involved in this process. Unlike polymerases that are responsible for DNA replication, error-prone DNA polymerases copy DNA with low fidelity leading to variations in immunoglobulin DNA sequences. Chronic lymphocytic leukemia (CLL) has been classified into mutated and unmutated subtypes based on the mutation status of the IGH variable region (IgVH). The latter has been associated with rapid disease progression and unfavorable outcome. Since determination of the IgVH mutations rely on direct sequencing of multiple PCR products and complex data analysis, the assay, although clinically useful, is not provided by many clinical laboratories. To determine whether error-prone DNA polymerases can be used as surrogate markers for IgVH mutation analysis, we examined the levels of several of them to see whether higher levels are associated with higher degree of IgVH mutation. We analyzed 30 CLL samples, of which 18 were unmutated and 12 were mutated. Quantitative PCR was performed to determine the expression levels of error-prone DNA polymerases mu, eta, iota, lambda and zeta. as well as DNA editing enzymes, terminal deoxynucleotidyl transferase (TdT) and activation-induced cytidine deaminase (AID). Statistical analysis revealed no differences in the levels of mRNA expression of any of these seven enzymes between mutated and unmutated cases of CLL. In addition, no differences were found between ZAP-70 positive and negative subgroups. The expression levels of the seven enzymes were then compared among CLL, follicular lymphomas (FL, n=8) and diffuse large B cell lymphomas (DLBCL, n=8). FL and DLBCL, being post-germinal center lymphomas that have undergone SHM, are expected to express higher levels of these enzymes than CLL. However, the statistical analysis revealed that CLL has significantly higher amount of mu, eta, iota, lambda, zeta and TdT expression than DLBCL. CLL also has significantly higher expression of iota, lambda, zeta and TdT than FL. In conclusion, our results suggest that DNA polymerases studied cannot be used as surrogate markers for IgVH analysis. Contrary to the common belief, the mRNA levels of DNA polymerases/DNA editing enzymes do not reflect the SHM activity in different B cell malignancies.than FL. In conclusion, our results suggest that DNA polymerases studied cannot be used as surrogate markers for IgVH analysis. Contrary to the common belief, the mRNA levels of DNA polymerases/DNA editing enzymes do not reflect the SHM activity in different B cell malignancies.

Malignant B Cells from Hairy Cell Leukemia Express an Innate Phenotype and Undergo IgD Class Switching in Response to Innate Environmental Factors, Including BAFF and APRIL.

Hairy cell leukemia (HCL) is a rare chronic lymphoproliferative disorder characterized by massive infiltration of the spleen by malignant B cells displaying a distinctive “hairy” morphology. These hairy cells (HCs) often contain hypermutated Ig genes, suggesting an ontogenetic relationship with post-germinal center memory B cells. However, unlike memory B cells, HCs usually express CD11c and CD103, two molecules typically associated with mucosal innate immune cells. The goal of this study was to elucidate the ontogeny and growth mechanisms of HCs. We found that HCs interacted with splenic endothelial cells expressing B cell-activating factor of the TNF family (BAFF) and a proliferation-inducing ligand (APRIL), two innate B cell-stimulating factors involved in T cell-independent antibody responses, including Ig class switching. In agreement with prior studies indicating that BAFF and APRIL play an important role in B cell tumors, HCs expressed transmembrane activator and calcium modulator and cyclophylin ligand interactor (TACI), B cell maturation antigen (BCMA), and BAFF receptor (BAFF-R), three receptors that bind BAFF and APRIL. In addition, HCs expressed microbial carbohydrate and endocytic receptors usually expressed by innate immune cells, including DEC-205 (CD205), mannose receptor (CD206), langerin (CD207) and dendritic cell-specific ICAM-3-grabbing non-integrin (CD209). This phenotype was different from that of naïve, germinal center, memory and plasmacytoid B cells, but similar to that of a subset of splenic marginal zone and mucosal B cells. Like these innate B cells, HCs contained activation-induced cytidine deaminase, a DNA-editing enzyme required for class switching, and showed signs of ongoing IgM-to-IgD class switching, a mucosal Ig diversifying process typically triggered by BAFF and APRIL. Our findings suggest that HCL may emerge as a result of chronic stimulation of an innate B cell precursor through a T-cell independent pathway involving microbial carbohydrates, BAFF and APRIL.

Long-Distance Tunneling Nanotubules Shuttle Viral Immunoglobulin Class Switch-Suppressing Factors from HIV-Infected Macrophages to B Cells.

Class switching from immunoglobulin M (IgM) to IgG, IgA or IgE is central to immunity against viruses and requires activation of B cells by CD4+ T cells through CD40 ligand (CD40L). Human immunodeficiency virus (HIV) evades protective T cell-dependent IgG and IgA responses against viral proteins and vaccines through mechanisms that remain unknown. Recent findings from our lab suggested the involvement of negative factor (Nef), an HIV accessory protein that suppresses CD40 signaling in B cells. The present work addressed the mechanism by which Nef invades B cells. Fluorescence microscopy-based studies showed that accumulation of Nef in infected lymphoid follicles was associated with severe loss of activation-induced cytidine deaminase (AID), a Nef-targeted DNA-editing enzyme essential for class switching. Loss of follicular AID correlated with in vivo inactivation of CD40 signaling and formation of long-distance membrane tethers connecting Nef-positive B cells with HIV-infected macrophages. By combining confocal microscopy, videomicroscopy and electron microscopy with standard molecular biology techniques, we demonstrated that macrophages harboring HIV or expressing a chimeric Nef-green-fluorescent protein were capable of transferring Nef-containing cargos to B cells via long-distance, actin-propelled and energy-dependent membrane channels, including tunneling nanotubules. Transfer of Nef to B cells involved a clathrin-dependent pathway that required the guanine nucleotide exchange factor Vav as well as downstream Rho, Rac and Ccd42 small GTPases. This pathway led to Nef-mediated inhibition of CD40-induced AID expression and IgG and IgA class switching. In summary, our findings indicate that HIV evades T cell-dependent IgG and IgA responses by shuttling virus-encoded Ig class switch-suppressing factors from infected macrophages to non-infected B cells via long-range membrane tethers, including tunneling nanotubules.

Target Cell APOBEC3C Can Induce Limited G-to-A Mutation in HIV-1

The evolutionary success of primate lentiviruses reflects their high capacity to mutate and adapt to new host species, immune responses within individual hosts, and, in recent years, antiviral drugs. APOBEC3G (A3G) and APOBEC3F (A3F) are host cell DNA-editing enzymes that induce extensive HIV-1 mutation that severely attenuates viral replication. The HIV-1 virion infectivity factor (Vif), expressed in vivo, counteracts the antiviral activity of A3G and A3F by inducing their degradation. Other APOBECs may contribute more to viral diversity by inducing less extensive mutations allowing viral replication to persist. Here we show that in APOBEC3C (A3C)-expressing cells infected with the patient-derived HIV-1 molecular clones 210WW, 210WM, 210MW, and 210MM, and the lab-adapted molecular clone LAI, viral G-to-A mutations were detected in the presence of Vif expression. Mutations occurred primarily in the GA context and were relatively infrequent, thereby allowing for spreading infection. The mutations were absent in cells lacking A3C but were induced after transient expression of A3C in the infected target cell. Inhibiting endogenous A3C by RNA interference in Magi cells prevented the viral mutations. Thus, A3C is necessary and sufficient for G-to-A mutations in some HIV-1 strains. A3C-induced mutations occur at levels that allow replication to persist and may therefore contribute to viral diversity. Developing drugs that inhibit A3C may be a novel strategy for delaying viral escape from immune or antiretroviral inhibition.

RNA and DNA Binding Properties of HIV-1 Vif Protein: A FLUORESCENCE STUDY

The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Some "non-permissive" cell lines cannot sustain replication of Vif(-) HIV-1 virions. In these cells, Vif counteracts the natural antiretroviral activity of the DNA-editing enzymes APOBEC3G/3F. Moreover, Vif is packaged into viral particles through a strong interaction with genomic RNA in viral nucleoprotein complexes. To gain insights into determinants of this binding process, we performed the first characterization of Vif/nucleic acid interactions using Vif intrinsic fluorescence. We determined the affinity of Vif for RNA fragments corresponding to various regions of the HIV-1 genome. Our results demonstrated preferential and moderately cooperative binding for RNAs corresponding to the 5'-untranslated region of HIV-1 (5'-untranslated region) and gag (cooperativity parameter omega approximately 65-80, and K(d) = 45-55 nM). In addition, fluorescence spectroscopy allowed us to point out the TAR apical loop and a short region in gag as primary strong affinity binding sites (K(d) = 9.5-14 nM). Interestingly, beside its RNA binding properties, the Vif protein can also bind the corresponding DNA oligonucleotides and their complementary counterparts with an affinity similar to the one observed for the RNA sequences, while other DNA sequences displayed reduced affinity. Taken together, our results suggest that Vif binding to RNA and DNA offers several non-exclusive ways to counteract APOBEC3G/3F factors, in addition to the well documented Vif-induced degradation by the proteasome and to the Vif-mediated repression of translation of these antiviral factors.

Epithelial cells trigger frontline immunoglobulin class switching through an SLPI-regulated pathway (41.7)

Abstract Epithelial cells (ECs) recruit B cells expressing class-switched IgG and IgA into mucosal effector sites and transport IgG and IgA antibodies across mucous membranes. Whether ECs initiate IgG and IgA class switching remains unknown. We found that ECs lining tonsillar crypts formed pockets populated by B cells expressing activation-induced cytidine deaminase (AID), a DNA-editing enzyme associated with ongoing class switch DNA recombination (CSR). ECs released AID- and CSR-inducing factors, such as B cell-activating factor of the TNF family (BAFF), after sensing microbial products through Toll-like receptors (TLRs). EC-induced IgG and IgA CSR was further amplified by thymic stromal lymphopoietin (TSLP), an epithelial IL-7-like cytokine that enhanced BAFF production by dendritic cells. Conversely, EC-induced IgG and IgA CSR was restrained by secretory leukocyte protease inhibitor (SLPI), an epithelial homeostatic protein that inhibited BAFF signaling in B cells. Our findings indicate that ECs function as mucosal guardians that orchestrate frontline IgG and IgA class switching through a TLR-inducible SLPI-regulated signaling program. This work was supported by NIH grants AI057653 (to A.C.) and by NIH T32 training grant AI07621 (to W. X.).

Activation Induced Cytidine Deaminase (AID) Is Required for Germinal-Center Derived Lymphomagenesis.

Most B cell non-Hodgkin's lymphomas (B-NHL) derive from germinal center (GC) B cells and their pathogenesis is associated with the accumulation of distinct genetic lesions, including chromosomal translocations and a more recently identified mechanism of genomic instability, termed aberrant somatic hypermutation. These alterations are thought to be due to mistakes occurring during two GC-associated immunoglobulin (Ig) genes remodeling processes: class switch recombination (CSR) and somatic hypermutation (SHM). However, this model has never been formally proven. To conclusively investigate the role of CSR and SHM in the pathogenesis of B-NHL, we examined whether lymphoma development in mice requires the function of activation induced cytidine deaminase (AID), a DNA editing enzyme expressed specifically in GC and activated B cells and essential for both processes. Three transgenic mouse models were generated by crossing lymphoma-prone mice (λMYC, λMYC/IμHABCL6 and IμHABCL6) with mice (AID−/−) that are unable to undergo both SHM and CSR. The λMYC mice develop a diffusely infiltrating monoclonal proliferation of pre-GC origin, with unmutated IgV genes and lack of BCL6 expression, and therefore presumably independent from AID-associated DNA remodeling events. Conversely, lymphomas in λMYC/IμHABCL6 and IμHABCL6 mice recapitulate GC/post GC-derived malignancies, in that the former display somatically mutated IgV genes and upregulation of post-GC markers (CD138) in most of the cases, while the latter develop a splenic lymphoproliferative syndrome that culminates, past 12 months of age, in clonal B cell lymphomas with DLBCL morphology and somatically mutated IgV genes (~70% of the animals) (Cattoretti et al., Cancer Cell 7:445–455, 2005). Mice were monitored for tumor incidence and survival, and a combination of histologic, immunophenotypic and gene expression profiling analysis was used for tumor characterization. As expected, no significant differences in event-free survival and lymphoma type were observed between AID-proficient and AID-deficient λMYC mice, in agreement with their pre-GC derivation. Conversely, a phenotypic shift of the tumor was observed in λMYC/IμHABCL6 mice when bred into an AID−/− background, with >80% of the cases (N=21/26) reverting to a pre-GC phenotype (loss of GC/post GC markers) undistinguishable from that of the λMYC and λMYC/AID−/− mice. Gene expression profile analysis on representative cases (N=10 λMYC/IμHABCL6 and 5 each for λMYC, λMYC/AIDKO, λMYC/IμHABCL6/AIDKO) confirmed significant phenotypic similarities between pre-GC derived λMYC lymphomas and the λMYC/IμHABCL6/AID −/− lymphomas, which co-segregated in a separate cluster from λMYC/IμHABCL6 tumors. Analogously, a significant reduction in DLBCL frequency was observed in the IμHABCL6/AIDKO cohort as compared to IμHABCL6 mice (N= 4/19, 21% vs 8/14, 57%; p=0.03).Taken together, these results indicate that GC-derived lymphomas cannot develop in the absence of AID, thereby providing direct support to the notion that AID-mediated mistakes in antigen receptor gene modification events (CSR and SHM) represent major contributors to B-NHL pathogenesis.

Mucosal Epithelial Cells Initiate Frontline Immunoglobulin Class Switching through an SLPI-Regulated Pathway.

Introduction. Class switching from IgM to IgG and IgA is central to immunity against microbes and usually occurs in draining lymph nodes and requires activation of B cells by CD4+ T cells expressing CD40 ligand. Growing evidence indicates that B cells can mount frontline IgG and IgA responses at mucosal sites of entry through an alternative CD40-independent pathway involving B cell-activating factor of the TNF family (BAFF, also known as BLyS) and a proliferation-inducing ligand (APRIL). These innate factors are usually produced by dendritic cells and stimulate B cells through at least three distinct receptors. Together with dendritic cells, epithelial cells have a key position at the host-environment interface. Therefore, we asked whether epithelial cells play a role in frontline antibody production.Methods. Tonsillar tissue sections from healthy donors were analyzed for expression of activation-induced cytidine deaminase (AID) by immunohistochemistry and in situ hybridization. A simplified in vitro model reproducing the geometry of mucosal surfaces was used to evaluate the role of epithelial cells in class switching. Briefly, primary epithelial cells and B cells were cultured in the upper and lower chambers, respectively, of a trans-well system. Monocyte-derived dendritic cells were positioned on a filter separating the two chambers. Various microbial product analogues were used to mimic infection. RNA interference was performed to knockdown BAFF in epithelial cells. AID expression, CSR, antibody production and signaling were evaluated in B cells as reported (Litinsky et al., Nat. Immunol. 2002, 3:822–829; Qiao et al., Nat. Immunol. 2006, 7:302–310).Results. We found that the upper respiratory mucosa of healthy subjects comprised intraepithelial pockets filled with B cells expressing AID, a DNA-editing enzyme associated with ongoing class switch DNA recombination (CSR). Epithelial cells released innate class switch-inducing factors, including BAFF, after sensing microbial products through TLRs, thereby inducing AID expression, CSR, and ultimately IgG and IgA production in neighboring B cells. Epithelial cell-induced antibodies comprised polyreactive IgG and IgA capable of recognizing multiple microbial determinants. Intraepithelial class switching was enhanced by thymic stromal lymphopoietin (TSLP), an epithelial IL-7-like cytokine that augments the innate B cell-licensing functions of dendritic cells, and restrained by secretory leukocyte protease inhibitor (SLPI), an epithelial alarm antiprotease that suppresses AID expression in activated B cells.Conclusions. The present findings indicate that epithelial cells function as non-immune sentinels capable to autonomously orchestrate compartmentalized IgG and IgA responses at the interface between host and environment. This implies that mucosal vaccines should activate both epithelial and immune cells to elicit optimal antibody production.

HIV accomplices and adversaries in macrophage infection

Cell surface and intracellular proteins in macrophages influence various steps in the life cycle of lentiviruses. Characterization of these restriction and/or cofactors is essential to understanding how macrophages become unwitting HIV hosts and in fact, can coexist with a heavy viral burden. Although many of the cellular pathways co-opted by HIV in macrophages mimic those seen in CD4+ T cells, emerging evidence reveals cellular constituents of the macrophage, which may be uniquely usurped by HIV. For example, in addition to CD4 and CCR5, membrane annexin II facilitates early steps in infection of macrophages, but not in T cells. Blockade of this pathway effectively diminishes macrophage infection. Viral binding engages a macrophage-centric signaling pathway and a transcriptional profile, including genes such as p21, which benefit the virus. Once inside the cell, multiple host cell molecules are engaged to facilitate virus replication and assembly. Although the macrophage is an enabler, it also possesses innate antiviral mechanisms, including apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3) family DNA-editing enzymes to inhibit replication of HIV. Differential expression of these enzymes, which are largely neutralized by HIV to protect its rebirth, is associated with resistance or susceptibility to the virus. Higher levels of the cytidine deaminases endow potential HIV targets with a viral shield, and IFN-alpha, a natural inducer of macrophage APOBEC expression, renders macrophages tougher combatants to HIV infection. These and other manipulatable pathways may give the macrophage a fighting chance in its battle against the virus.

The TNF Family Members BAFF and APRIL Play an Important Role in Hodgkin Lymphoma.

Introduction. B cell-activating factor of the TNF family (BAFF) and a proliferation-inducing ligand (APRIL), a BAFF-related molecule, play a key role in the survival and proliferation of mature B cells. In addition, BAFF and APRIL cooperate with IL-4 to induce class switch DNA recombination (CSR) from IgM (or IgG) to IgG, IgA or IgE. This process requires activation-induced-cytidine deaminase (AID), a DNA-editing enzyme involved also in Ig somatic hypermutation and lymphomagenesis. BAFF and APRIL are usually produced by myeloid cells, including dendritic cells, macrophages and granulocytes, and engage three receptors preferentially expressed on B cells, including transmembrane activator and calcium modulator and cyclophylin ligand interactor (TACI), B cell maturation antigen (BCMA), and BAFF receptor (BAFF-R). Our previous studies show that BAFF and APRIL are EBV-inducible molecules implicated in B cell non-Hodgkin's lymphoma (NHL). The scope of the present studies was to elucidate the expression and function of BAFF, APRIL, TACI, BCMA and BAFF-R in Hodgkin lymphoma (HL).Methods. Tissue sections from 5 primary EBV+ HL cases and 5 primary EBV− HL cases were analyzed for BAFF, APRIL, TACI, BCMA, and BAFF-R expression through immunohistochemistry. RS cells from 6 primary cases were microdissected and analyzed for the expression of AID and CSR byproducts by RT-PCR. The expression of BAFF, APRIL, TACI, BCMA, BAFF-R, AID, and CSR byproducts was also analyzed in 5 HL cell lines cultured in the presence or absence of recombinant BAFF, APRIL and cytokines as previously described1,2,3.Results. We found that the reactive infiltrate of primary HL tumors comprises non-malignant elements, such as macrophages, granulocytes and plasma cells, expressing BAFF and APRIL. Also a variable proportion of malignant CD30+ Reed-Sternberg (RS) cells from both EBV+ and EBV− HL cases express BAFF and APRIL. Unlike NHL B cells, which usually express BAFF-R, primary RS cells and RS cell lines lack BAFF-R, but express TACI and BCMA. In the presence of BAFF or APRIL, RS cell lines are rescued from spontaneous or induced apoptosis. This effect is associated with activation of NF-κB through a classical pathway. Increased RS cell survival is also associated with up-regulation of the pro-survival BCL-2 and BCL-XL proteins, and down-regulation of the pro-apoptotic BAX protein. Finally, in the presence of BAFF or APRIL and IL-4, RS cell lines up-regulate AID expression and increase their spontaneous CSR activity. Of note, AID expression extends to primary RS cells and is associated with ongoing CSR.Conclusions. Our studies indicate that BAFF and APRIL stimulate malignant RS cells through both autocrine and paracrine pathways. Engagement of TACI and BCMA receptors by BAFF and APRIL may enhance the expansion of RS cells by attenuating apoptosis through a mechanism involving NF-κB and BCL family proteins. By up-regulating AID, signals emanating from TACI and BCMA receptors might also introduce genomic instability. Finally, considering that TACI, BCMA and AID are B cell-specific molecules and that CSR is a process confined to B cells, our findings consolidate the notion that RS cells derive from a B cell precursor.

Ancient adaptive evolution of the primate antiviral DNA-editing enzyme APOBEC3G.

Host genomes have adopted several strategies to curb the proliferation of transposable elements and viruses. A recently discovered novel primate defense against retroviral infection involves a single-stranded DNA-editing enzyme, APOBEC3G, that causes hypermutation of HIV. The HIV-encoded virion infectivity factor (Vif) protein targets APOBEC3G for destruction, setting up a genetic conflict between the APOBEC3G and Vif genes. This kind of conflict leads to rapid fixation of mutations that alter amino acids at the protein–protein interface, referred to as positive selection. We show that the APOBEC3G gene has been subject to strong positive selection throughout the history of primate evolution. Unexpectedly, this selection appears more ancient than, and is likely only partially caused by, modern lentiviruses. Furthermore, five additional APOBEC genes in the human genome appear to be engaged in similar genetic conflicts, displaying some of the highest signals for positive selection in the human genome. Despite being only recently discovered, editing of RNA and DNA may thus represent an ancient form of host defense in primate genomes.

A second human antiretroviral factor, APOBEC3F, is suppressed by the HIV-1 and HIV-2 Vif proteins.

The HIV-1 Vif protein suppresses the inhibition of viral replication caused by the human antiretroviral factor APOBEC3G. As a result, HIV-1 mutants that do not express the Vif protein are replication incompetent in 'nonpermissive' cells, such as primary T cells and the T-cell line CEM, that express APOBEC3G. In contrast, Vif-defective HIV-1 replicates effectively in 'permissive' cell lines, such as a derivative of CEM termed CEM-SS, that do not express APOBEC3G. Here, we show that a second human protein, APOBEC3F, is also specifically packaged into HIV-1 virions and inhibits their infectivity. APOBEC3F binds the HIV-1 Vif protein specifically and Vif suppresses both the inhibition of virus infectivity caused by APOBEC3F and virion incorporation of APOBEC3F. Surprisingly, APOBEC3F and APOBEC3G are extensively coexpressed in nonpermissive human cells, including primary lymphocytes and the cell line CEM, where they form heterodimers. In contrast, both genes are quiescent in the permissive CEM derivative CEM-SS. Together, these data argue that HIV-1 Vif has evolved to suppress at least two distinct but related human antiretroviral DNA-editing enzymes.

Hyper-immunoglobulin-M syndromes caused by an intrinsic B cell defect.

Elucidation of the molecular basis of hyper-immunoglobulin-M syndromes has provided considerable insight into the molecular events involved in antibody maturation, including immunoglobulin class switch recombination and the generation of somatic hypermutation. The identification of activation-induced cytidine deaminase deficiency (hyper-immunoglobulin-M syndrome 2) has revealed the key role played by this inducible B cell-specific molecule in both class switch recombination and somatic hypermutation. Data from Escherichia coli and in-vitro assays have strongly suggested that activation-induced cytidine deaminase acts as a DNA-editing enzyme in these processes. The recent description of a new hyper-immunoglobulin-M syndrome caused by mutations in the gene encoding the uracil-N glycosylase provided further evidence that activation-induced cytidine deaminase acts on deoxycytidine in the switch and variable regions. Indeed, uracil-N glycosylase is required to remove the uracil residues integrated into DNA following deoxycytidine deamination by activation-induced cytidine deaminase. Another hyper-immunoglobulin-M condition has recently been described (hyper-immunoglobulin-M syndrome 4). Its molecular basis is unknown, but it appears to be a homogeneous entity characterized by an intrinsic B cell defective class switch recombination but normal generation of somatic hypermutation. It is probably caused by a class switch recombination-specific DNA repair defect because class switch recombination-induced DNA breaks in S regions are normally detected in patients with this condition. The heterogeneity in hyper-immunoglobulin-M syndromes will continue to shed light on the molecular mechanisms of class switch recombination and somatic hypermutation. The description of hyper-immunoglobulin-M syndromes may therefore lead to improvements in the care of these patients.

Oxygen Free Radical Damage to DNA: TRANSLESION SYNTHESIS BY HUMAN DNA POLYMERASE η AND RESISTANCE TO EXONUCLEASE ACTION AT CYCLOPURINE DEOXYNUCLEOSIDE RESIDUES

Cyclopurine deoxynucleosides are common DNA lesions generated by exposure to reactive oxygen species under hypoxic conditions. The S and R diastereoisomers of cyclodeoxyadenosine on DNA were investigated separately for their ability to block 3' to 5' exonucleases. The mammalian DNA-editing enzyme DNase III (TREX1) was blocked by both diastereoisomers, whereas only the S diastereoisomer was highly efficient in preventing digestion by the exonuclease function of T4 DNA polymerase. Digestion in both cases was frequently blocked one residue before the modified base. Oligodeoxyribonucleotides containing a cyclodeoxyadenosine residue were further employed as templates for synthesis by human DNA polymerase eta (pol eta). pol eta could catalyze translesion synthesis on the R diastereoisomer of cyclodeoxyadenosine. On the S diastereoisomer, pol eta could catalyze the incorporation of one nucleotide opposite the lesion but could not continue elongation. Although pol eta preferentially incorporated dAMP opposite the R diastereoisomer, elongation continued only when dTMP was incorporated, suggesting bypass of this lesion by pol eta with reasonable fidelity. With the S diastereoisomer, pol eta mainly incorporated dAMP or dTMP opposite the lesion but could not elongate even after incorporating a correct nucleotide. These data suggest that the S diastereoisomer may be a more cytotoxic DNA lesion than the R diastereoisomer.

A human DNA editing enzyme homologous to the Escherichia coli DnaQ/MutD protein.

Mammalian DNA polymerases alpha and beta lack 3' exonuclease activity and are unable to edit errors after DNA synthesis. However, editing exonucleases can be functions of separate polypeptides. We isolated a widely distributed DNA-specific 3' exonuclease from rabbit liver nuclei, sequenced tryptic peptides by mass spectrometry, and identified the corresponding human open reading frame. The protein expressed from the cloned human sequence exhibits 3' exonuclease activity. The human clone shares sequence homology with the editing function of the Escherichia coli DNA polymerase III holoenzyme, i.e., the DnaQ/MutD protein, and weakly with the editing 3' exonuclease domain of eukaryotic DNA polymerase epsilon. The gene maps to human chromosome 3p21.2-21.3. In a reconstituted human DNA repair system containing DNA polymerase beta and DNA ligase III-XRCC1, accurate rejoining of a 3' mismatched base residue at a single-strand break is dependent on addition of the exonuclease.