SUMMARY Class switch recombination (CSR) requires activation-induced deaminase (AID) to instigate double-stranded DNA breaks at the immunoglobulin locus. DNA breaks activate the DNA damage response (DDR) by inducing phosphorylation of histone H2AX followed by non-homologous end joining (NHEJ) repair. We carried out a genome-wide screen to identify CSR factors. We found that Usp22, Eny2, and Atxn7, members of the Spt-Ada-Gcn5-acetyltransferase (SAGA) deubiquitination module, are required for deubiquitination of H2BK120ub following DNA damage, are critical for CSR, and function downstream of AID. The SAGA deubiquitinase activity was required for optimal irradiation-induced γH2AX formation, and failure to remove H2BK120ub inhibits ATM- and DNAPK-induced γH2AX formation. Consistent with this effect, these proteins were found to function upstream of various double-stranded DNA repair pathways. This report demonstrates that deubiquitination of histone H2B impacts the early stages of the DDR and is required for the DNA repair phase of CSR.

Antibody diversification necessitates targeted mutation of regions within the immunoglobulin locus by activation-induced cytidine deaminase (AID). While AID is known to act on single-stranded DNA (ssDNA), the source, structure, and distribution of these substrates in vivo remain unclear. Using the technique of in situ bisulfite treatment, we characterized these substrates—which we found to be unique to actively transcribed genes—as short ssDNA regions, that are equally distributed on both DNA strands. We found that the frequencies of these ssDNA patches act as accurate predictors of AID activity at reporter genes in hypermutating and class switching B cells as well as in Escherichia coli. Importantly, these ssDNA patches rely on transcription, and we report that transcription-induced negative supercoiling enhances both ssDNA tract formation and AID mutagenesis. In addition, RNaseH1 expression does not impact the formation of these ssDNA tracts indicating that these structures are distinct from R-loops. These data emphasize the notion that these transcription-generated ssDNA tracts are one of many in vivo substrates for AID.

An effective immune response requires B cells to produce several classes of antibodies through the process of class switch recombination (CSR). Activation-induced cytidine deaminase initiates CSR by deaminating deoxycytidines at switch regions within the Ig locus. This activity leads to double-stranded DNA break formation at the donor and recipient switch regions that are subsequently synapsed and ligated in a 53BP1-dependent process that remains poorly understood. The DNA damage response E3 ubiquitin ligases RNF8 and RNF168 were recently shown to facilitate recruitment of 53BP1 to sites of DNA damage. Here we show that the ubiquitination pathway mediated by RNF8 and RNF168 plays an integral part in CSR. Using the CH12F3-2 mouse B cell line that undergoes CSR to IgA at high rates, we demonstrate that knockdown of RNF8, RNF168, and 53BP1 leads to a significant decrease in CSR. We also show that 53BP1-deficient CH12F3-2 cells are protected from apoptosis mediated by the MDM2 inhibitor Nutlin-3. In contrast, deficiency in either E3 ubiquitin ligase does not protect cells from Nutlin-3-mediated apoptosis, indicating that RNF8 and RNF168 do not regulate all functions of 53BP1.53BP1 | activation-induced cytidine deaminase | DNA damage response P art of an effective immune response requires the production of antibodies of different classes, each of which mediates a different effector function. This process is initiated by activationinduced cytidine deaminase (AID), which induces class switch recombination (CSR) by deaminating deoxycytidines within Ig switch regions (1, 2). The recognition and subsequent processing of the mutated residues by base excision repair and/or mismatch repair machineries generates double-stranded DNA breaks (DSBs) at switch regions (3). In an attempt to mend the ensuing breaks, B cells mount a damage response similar to that signaled by irradiated cells (1). Ultimately, AID-induced DSBs are repaired predominantly by nonhomologous end-joining (4, 5) and to a lesser extent by an alternative end-joining pathway (6).Generally, DSBs are readily recognized by sensor-kinase protein complexes, including the Mre11-Rad50-Nbs1 (MRN)/ataxiatelangiectasia mutated (ATM), Ku70/Ku80-DNA-PKcs, and ataxiatelangiectasia and Rad3-related (ATR)-ATR-interacting protein complexes (7). Upon binding to a DSB site, these factors are activated to trigger a cascade of events that culminates in cell cycle delay and/or DNA repair (7). During this process, sequential chromatin modifications around the break sites appear to be crucial for adequate resolution of the DNA breaks. Active chromatin modification is initiated by ATM-dependent phosphorylation of the histone variant H2AX to the γH2AX form (8-10). γ-H2AX then preferentially binds the tandem-BRCA1 C-terminal domain (BRCT) motifs of mediator of DNA damage checkpoint 1 (MDC1) (11, 12), which in turn recruits more MRN/ATM, thereby amplifying the γ-H2AX signal (11, 12). Recently, it was shown that ATM also phosphorylates MDC1 at a TQxF consensus, allowing for the recruitmen...

DNA breaks caused by recombination-activating gene 1 (RAG1) and activation-induced cytidine deaminase (AID) induce c-myc/immunoglobulin (Ig) heavy chain chromosomal translocations and thereby stimulate lymphomagenesis. However, constitutive expression of c-myc alone is not sufficient to induce lymphomas. Because RAG1 and AID activity occurs outside of Ig genes, we assessed whether these enzymes provide the secondary genetic lesions in El c-myc transgenic mice to promote lymphoma development. We found that the tumor incidence and tumor phenotype in El c-myc transgenic mice is similar in AID þ / þ , AID þ /À and AID À/À backgrounds in both specific pathogen-free and conventional animal facilities, indicating that AID does not contribute to lymphoma development in El c-myc transgenic mice. To examine the role of RAG proteins in El c-myc mice, we examined El c-myc transgenic mice that harbor the Ig-HEL heavy-and light-chain transgenes, and thus have reduced RAG expression in B cells. We found that tumor incidence was not affected by these Ig transgenes. However, we found that RAG1 À/À El c-myc mice exhibited accelerated tumor development compared to controls. This data combined with our finding that El c-myc mice lived longer in the conventional facility than in the specific pathogen-free facility suggest an immunemediated activity that suppresses lymphoma development.

C-Myc is one of the most common targets of genetic alterations in human cancers. Although overexpression of c-Myc in the B cell compartment predisposes to lymphomas, secondary mutations are required for disease manifestation. In this article, we show that genetic deficiencies causing arrested B cell development and accumulation of B cell progenitors lead to accelerated lymphomagenesis in E c-myc transgenic mice. This result suggests that B cell progenitors are more prone than their mature counterparts to developing secondary oncogenic lesions that complement c-Myc in promoting transformation. To investigate the nature of these oncogenic lesions, we examined E c-myc mice deficient in mismatch repair function. We report that Msh2 ؊/؊ E c-myc and Msh2 G674A/G674A E c-myc mice rapidly succumb to pro-B cell stage lymphomas, indicating that Msh2-dependent mismatch repair function actively suppresses c-Myc-associated oncogenesis during early B cell development.apoptosis ͉ mismatch repair ͉ oncogenesis ͉ p53

Mycobacterium tuberculosis-infected macrophages demonstrate diminished capacity to present antigens via class II MHC molecules. Since successful class II MHC-restricted antigen presentation relies on the actions of endocytic proteases, we asked whether the activities of cathepsins (Cat) B, S and L-three major lysosomal cysteine proteases-are modulated in macrophages infected with pathogenic Mycobacterium spp. Infection of murine bone marrow-derived macrophages with either Mycobacterium avium or M. tuberculosis had no obvious effect on Cat B or Cat S activity. In contrast, the activity of Cat L was altered in infected cells. Specifically, whereas the 24-kDa two-chain mature form of active Cat L predominated in uninfected cells, we observed an increase in the steady-state activity of the precursor single-chain (30 kDa) and 25-kDa two-chain forms of the enzyme in cells infected with either M. avium or M. tuberculosis. Pulse-chase analyses revealed that maturation of nascent, single-chain Cat L into the 25-kDa two-chain form was impaired in infected macrophages, and that maturation into the 24-kDa two-chain form did not occur. Consistent with these data, M. avium infection inhibited the IFNgamma-induced secretion of active two-chain Cat L by macrophages. Viable bacilli were not required to disrupt Cat L maturation, suggesting that a constitutively expressed mycobacterial component was responsible. The absence of the major active form of lysosomal Cat L in M. avium- and M. tuberculosis-infected macrophages may influence the types of T cell epitopes generated in these antigen-presenting cells, and/or the rate of class II MHC peptide loading.

IntroductionMismatch repair (MMR) is an evolutionary conserved DNA repair pathway that is required to repair mutations that arise through DNA replication or other processes. 1,2 MutS homologue 2 (Msh2) dimerizes with either Msh6 or Msh3 to recognize single base-pair mismatches or mismatches caused by insertion/deletions, respectively. 2 After binding to the mismatch, Msh2 hydrolyzes ATP, which induces a change in the heterodimer that allows for the recruitment of downstream repair factors such as MutL homologue 1, postmeiotic segregation 2, and exonuclease 1 (Exo1). 3,4 Because of Msh2's essential role in repairing DNA mutations, Msh2 Ϫ/Ϫ mice have 5-fold, 11-fold, and 15-fold greater mutation frequencies in the brain, small intestine, and thymus, respectively, 5 and are predisposed to cancer. [6][7][8] During an immune response, Ig genes undergo somatic hypermutation (SHM) to produce high-affinity antibodies. SHM is initiated by activation-induced cytidine deaminase (AID), 9 which deaminates cytidine molecules within the V region to produce dU:dG mispairs. [10][11][12] The dU:dG mismatch is recognized by the Msh2/Msh6 heterodimer, but instead of repairing this mutation, MMR produces mutations. [13][14][15][16][17][18] Mice deficient in Msh2, Msh6, postmeiotic segregation 2, MutL homologue 1, or Exo1 have an approximately 2-fold decrease in overall mutation frequency at the V region. 13,14,[16][17][18] In particular, there is an approximately10-fold decrease in mutations at A:T base pairs at the V region in MSH2-deficient mice, whereas the mutation frequency at G:C base pairs is unaltered. [15][16][17][18][19] These results indicate that the MMR pathway is required to produce mutations at A:T base pairs in the V region. MMR proteins have also been implicated in mutagenic processes that cause disease, including trinucleotide-repeat diseases. 20 Two sets of recent results suggest the possibility that the MMR system is generally defective in germinal center (GC) B cells.Ouchida et al 21 reported that GC B cells have a generally greater mutation rate than other cells, as measured by the frequency of mutations on a lacZ transgene. Liu et al 22 found that in Msh2 mutant mice, the mutation frequency of AID-sensitive genes was either lower or only slightly greater than in wild-type (WT) cells; they argued that the Msh2-dependent pathway was not functioning normally in GC cells. Together, these results would fit a simple unified model in which the expression of a GC/B cell-specific factor commandeers MMR and transforms this DNA repair pathway into a mutagenic pathway.An alternative explanation is that the MMR pathway is only mutagenic at sites mutated by AID, which would argue against a factor that disrupts global MMR function in GC B cells. To explore this issue, we examined the impact of Msh2-deficiency at a lacI transgene that is not mutated by AID in GC B cells. Our results indicate that Msh2 efficiently repairs lacI mutations in all cell populations examined, including GC B cells. Thus, MMR functions normally in GC ...

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