共查询到20条相似文献,搜索用时 31 毫秒
1.
Basu U Chaudhuri J Alpert C Dutt S Ranganath S Li G Schrum JP Manis JP Alt FW 《Nature》2005,438(7067):508-511
Antibodies, which are produced by B-lineage cells, consist of immunoglobulin heavy (IgH) and light (IgL) chains that have amino-terminal variable regions and carboxy-terminal constant regions. In response to antigens, B cells undergo two types of genomic alterations to increase antibody diversity. Affinity for antigen can be increased by introduction of point mutations into IgH and IgL variable regions by somatic hypermutation. In addition, antibody effector functions can be altered by changing the expressed IgH constant region exons through IgH class switch recombination (CSR). Somatic hypermutation and CSR both require the B-cell-specific activation-induced cytidine deaminase protein (AID), which initiates these reactions through its single-stranded (ss)DNA-specific cytidine deaminase activity. In biochemical assays, replication protein A (RPA), a ssDNA-binding protein, associates with phosphorylated AID from activated B cells and enhances AID activity on transcribed double-stranded (ds)DNA containing somatic hypermutation or CSR target sequences. This AID-RPA association, which requires phosphorylation, may provide a mechanism for allowing AID to access dsDNA targets in activated B cells. Here we show that AID from B cells is phosphorylated on a consensus protein kinase A (PKA) site and that PKA is the physiological AID kinase. Thus, AID from non-lymphoid cells can be functionally phosphorylated by recombinant PKA to allow interaction with RPA and promote deamination of transcribed dsDNA substrates. Moreover, mutation of the major PKA phosphorylation site of AID preserves ssDNA deamination activity, but markedly reduces RPA-dependent dsDNA deamination activity and severely impairs the ability of AID to effect CSR in vivo. We conclude that PKA has a critical role in post-translational regulation of AID activity in B cells. 相似文献
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AID is required to initiate Nbs1/gamma-H2AX focus formation and mutations at sites of class switching. 总被引:11,自引:0,他引:11
S Petersen R Casellas B Reina-San-Martin H T Chen M J Difilippantonio P C Wilson L Hanitsch A Celeste M Muramatsu D R Pilch C Redon T Ried W M Bonner T Honjo M C Nussenzweig A Nussenzweig 《Nature》2001,414(6864):660-665
Class switch recombination (CSR) is a region-specific DNA recombination reaction that replaces one immunoglobulin heavy-chain constant region (Ch) gene with another. This enables a single variable (V) region gene to be used in conjunction with different downstream Ch genes, each having a unique biological activity. The molecular mechanisms that mediate CSR have not been defined, but activation-induced cytidine deaminase (AID), a putative RNA-editing enzyme, is required for this reaction. Here we report that the Nijmegen breakage syndrome protein (Nbs1) and phosphorylated H2A histone family member X (gamma-H2AX, also known as gamma-H2afx), which facilitate DNA double-strand break (DSB) repair, form nuclear foci at the Ch region in the G1 phase of the cell cycle in cells undergoing CSR, and that switching is impaired in H2AX-/- mice. Localization of Nbs1 and gamma-H2AX to the Igh locus during CSR is dependent on AID. In addition, AID is required for induction of switch region (S mu)-specific DNA lesions that precede CSR. These results place AID function upstream of the DNA modifications that initiate CSR. 相似文献
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The AID enzyme induces class switch recombination in fibroblasts 总被引:14,自引:0,他引:14
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Human meiotic recombinase Dmc1 promotes ATP-dependent homologous DNA strand exchange 总被引:1,自引:0,他引:1
Homologous recombination is crucial for the repair of DNA breaks and maintenance of genome stability. In Escherichia coli, homologous recombination is dependent on the RecA protein. In the presence of ATP, RecA mediates the homologous DNA pairing and strand exchange reaction that links recombining DNA molecules. DNA joint formation is initiated through the nucleation of RecA onto single-stranded DNA (ssDNA) to form helical nucleoprotein filaments. Two RecA-like recombinases, Rad51 and Dmc1, exist in eukaryotes. Whereas Rad51 is needed for both mitotic and meiotic recombination events, the function of Dmc1 is restricted to meiosis. Here we examine human Dmc1 protein (hDmc1) for the ability to promote DNA strand exchange, and show that hDmc1 mediates strand exchange between paired DNA substrates over at least several thousand base pairs. DNA strand exchange requires ATP and is strongly dependent on the heterotrimeric ssDNA-binding molecule replication factor A (RPA). We present evidence that hDmc1-mediated DNA recombination initiates through the nucleation of hDmc1 onto ssDNA to form a helical nucleoprotein filament. The DNA strand exchange activity of hDmc1 is probably indispensable for repair of DNA double-strand breaks during meiosis and for maintaining the ploidy of meiotic chromosomes. 相似文献
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After gene rearrangement, immunoglobulin variable genes are diversified by somatic hypermutation or gene conversion, whereas the constant region is altered by class-switch recombination. All three processes depend on activation-induced cytidine deaminase (AID), a B-cell-specific protein that has been proposed (because of sequence homology) to function by RNA editing. But indications that the three gene diversification processes might be initiated by a common type of DNA lesion, together with the proposal that there is a first phase of hypermutation that targets dC/dG, suggested to us that AID may function directly at dC/dG pairs. Here we show that expression of AID in Escherichia coli gives a mutator phenotype that yields nucleotide transitions at dC/dG in a context-dependent manner. Mutation triggered by AID is enhanced by a deficiency of uracil-DNA glycosylase, which indicates that AID functions by deaminating dC residues in DNA. We propose that diversification of functional immunoglobulin genes is triggered by AID-mediated deamination of dC residues in the immunoglobulin locus with the outcome--that is, hypermutation phases 1 and 2, gene conversion or switch recombination--dependent on the way in which the initiating dU/dG lesion is resolved. 相似文献
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Flynn RL Centore RC O'Sullivan RJ Rai R Tse A Songyang Z Chang S Karlseder J Zou L 《Nature》2011,471(7339):532-536
Maintenance of telomeres requires both DNA replication and telomere 'capping' by shelterin. These two processes use two single-stranded DNA (ssDNA)-binding proteins, replication protein A (RPA) and protection of telomeres 1 (POT1). Although RPA and POT1 each have a critical role at telomeres, how they function in concert is not clear. POT1 ablation leads to activation of the ataxia telangiectasia and Rad3-related (ATR) checkpoint kinase at telomeres, suggesting that POT1 antagonizes RPA binding to telomeric ssDNA. Unexpectedly, we found that purified POT1 and its functional partner TPP1 are unable to prevent RPA binding to telomeric ssDNA efficiently. In cell extracts, we identified a novel activity that specifically displaces RPA, but not POT1, from telomeric ssDNA. Using purified protein, here we show that the heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) recapitulates the RPA displacing activity. The RPA displacing activity is inhibited by the telomeric repeat-containing RNA (TERRA) in early S phase, but is then unleashed in late S phase when TERRA levels decline at telomeres. Interestingly, TERRA also promotes POT1 binding to telomeric ssDNA by removing hnRNPA1, suggesting that the re-accumulation of TERRA after S phase helps to complete the RPA-to-POT1 switch on telomeric ssDNA. Together, our data suggest that hnRNPA1, TERRA and POT1 act in concert to displace RPA from telomeric ssDNA after DNA replication, and promote telomere capping to preserve genomic integrity. 相似文献
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Ramiro AR Jankovic M Callen E Difilippantonio S Chen HT McBride KM Eisenreich TR Chen J Dickins RA Lowe SW Nussenzweig A Nussenzweig MC 《Nature》2006,440(7080):105-109
Chromosomal translocations involving the immunoglobulin switch region are a hallmark feature of B-cell malignancies. However, little is known about the molecular mechanism by which primary B cells acquire or guard against these lesions. Here we find that translocations between c-myc and the IgH locus (Igh) are induced in primary B cells within hours of expression of the catalytically active form of activation-induced cytidine deaminase (AID), an enzyme that deaminates cytosine to produce uracil in DNA. Translocation also requires uracil DNA glycosylase (UNG), which removes uracil from DNA to create abasic sites that are then processed to double-strand breaks. The pathway that mediates aberrant joining of c-myc and Igh differs from intrachromosomal repair during immunoglobulin class switch recombination in that it does not require histone H2AX, p53 binding protein 1 (53BP1) or the non-homologous end-joining protein Ku80. In addition, translocations are inhibited by the tumour suppressors ATM, Nbs1, p19 (Arf) and p53, which is consistent with activation of DNA damage- and oncogenic stress-induced checkpoints during physiological class switching. Finally, we demonstrate that accumulation of AID-dependent, IgH-associated chromosomal lesions is not sufficient to enhance c-myc-Igh translocations. Our findings reveal a pathway for surveillance and protection against AID-dependent DNA damage, leading to chromosomal translocations. 相似文献
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Activation-induced cytidine deaminase turns on somatic hypermutation in hybridomas 总被引:19,自引:0,他引:19
The production of high-affinity protective antibodies requires somatic hypermutation (SHM) of the antibody variable (V)-region genes. SHM is characterized by a high frequency of point mutations that occur only during the centroblast stage of B-cell differentiation. Activation-induced cytidine deaminase (AID), which is expressed specifically in germinal-centre centroblasts, is required for this process, but its exact role is unknown. Here we show that AID is required for SHM in the centroblast-like Ramos cells, and that expression of AID is sufficient to induce SHM in hybridoma cells, which represent a later stage of B-cell differentiation that does not normally undergo SHM. In one hybridoma, mutations were exclusively in G*C base pairs that were mostly within RGYW or WRCY motifs, suggesting that AID has primary responsibility for mutations at these nucleotides. The activation of SHM in hybridomas indicates that AID does not require other centroblast-specific cofactors to induce SHM, suggesting either that it functions alone or that the factors it requires are expressed at other stages of B-cell differentiation. 相似文献
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Liu M Duke JL Richter DJ Vinuesa CG Goodnow CC Kleinstein SH Schatz DG 《Nature》2008,451(7180):841-845
Somatic hypermutation introduces point mutations into immunoglobulin genes in germinal centre B cells during an immune response. The reaction is initiated by cytosine deamination by the activation-induced deaminase (AID) and completed by error-prone processing of the resulting uracils by mismatch and base excision repair factors. Somatic hypermutation represents a threat to genome integrity and it is not known how the B cell genome is protected from the mutagenic effects of somatic hypermutation nor how often these protective mechanisms fail. Here we show, by extensive sequencing of murine B cell genes, that the genome is protected by two distinct mechanisms: selective targeting of AID and gene-specific, high-fidelity repair of AID-generated uracils. Numerous genes linked to B cell tumorigenesis, including Myc, Pim1, Pax5, Ocab (also called Pou2af1), H2afx, Rhoh and Ebf1, are deaminated by AID but escape acquisition of most mutations through the combined action of mismatch and base excision repair. However, approximately 25% of expressed genes analysed were not fully protected by either mechanism and accumulated mutations in germinal centre B cells. Our results demonstrate that AID acts broadly on the genome, with the ultimate distribution of mutations determined by a balance between high-fidelity and error-prone DNA repair. 相似文献
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Human CtIP promotes DNA end resection 总被引:3,自引:0,他引:3
Sartori AA Lukas C Coates J Mistrik M Fu S Bartek J Baer R Lukas J Jackson SP 《Nature》2007,450(7169):509-514
In the S and G2 phases of the cell cycle, DNA double-strand breaks (DSBs) are processed into single-stranded DNA, triggering ATR-dependent checkpoint signalling and DSB repair by homologous recombination. Previous work has implicated the MRE11 complex in such DSB-processing events. Here, we show that the human CtIP (RBBP8) protein confers resistance to DSB-inducing agents and is recruited to DSBs exclusively in the S and G2 cell-cycle phases. Moreover, we reveal that CtIP is required for DSB resection, and thereby for recruitment of replication protein A (RPA) and the protein kinase ATR to DSBs, and for the ensuing ATR activation. Furthermore, we establish that CtIP physically and functionally interacts with the MRE11 complex, and that both CtIP and MRE11 are required for efficient homologous recombination. Finally, we reveal that CtIP has sequence homology with Sae2, which is involved in MRE11-dependent DSB processing in yeast. These findings establish evolutionarily conserved roles for CtIP-like proteins in controlling DSB resection, checkpoint signalling and homologous recombination. 相似文献
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Ira G Pellicioli A Balijja A Wang X Fiorani S Carotenuto W Liberi G Bressan D Wan L Hollingsworth NM Haber JE Foiani M 《Nature》2004,431(7011):1011-1017
A single double-strand break (DSB) induced by HO endonuclease triggers both repair by homologous recombination and activation of the Mec1-dependent DNA damage checkpoint in budding yeast. Here we report that DNA damage checkpoint activation by a DSB requires the cyclin-dependent kinase CDK1 (Cdc28) in budding yeast. CDK1 is also required for DSB-induced homologous recombination at any cell cycle stage. Inhibition of homologous recombination by using an analogue-sensitive CDK1 protein results in a compensatory increase in non-homologous end joining. CDK1 is required for efficient 5' to 3' resection of DSB ends and for the recruitment of both the single-stranded DNA-binding complex, RPA, and the Rad51 recombination protein. In contrast, Mre11 protein, part of the MRX complex, accumulates at unresected DSB ends. CDK1 is not required when the DNA damage checkpoint is initiated by lesions that are processed by nucleotide excision repair. Maintenance of the DSB-induced checkpoint requires continuing CDK1 activity that ensures continuing end resection. CDK1 is also important for a later step in homologous recombination, after strand invasion and before the initiation of new DNA synthesis. 相似文献
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Altering the pathway of immunoglobulin hypermutation by inhibiting uracil-DNA glycosylase 总被引:34,自引:0,他引:34
A functional immune system depends on the production of a wide range of immunoglobulin molecules. Immunoglobulin variable region (IgV) genes are diversified after gene rearrangement by hypermutation. In the DNA deamination model, we have proposed that deamination of dC residues to dU by activation-induced deaminase (AID) triggers this diversification. In hypermutating chicken DT40 B cells, most IgV mutations are dC --> dG/dA or dG --> dC/dT transversions, which are proposed to result from replication over sites of base loss produced by the excision activity of uracil-DNA glycosylase. Blocking the activity of uracil-DNA glycosylase should instead lead to replication over the dU lesion, resulting in dC --> dT (and dG --> dA) transitions. Here we show that expression in DT40 cells of a bacteriophage-encoded protein that inhibits uracil-DNA glycosylase shifts the pattern of IgV gene mutations from transversion dominance to transition dominance. This is good evidence that antibody diversification involves dC --> dU deamination within the immunoglobulin locus itself. 相似文献
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APOBEC-2 (APO2) belongs to the family of apolipoprotein B messenger RNA-editing enzyme catalytic (APOBEC) polypeptides, which deaminates mRNA and single-stranded DNA. Different APOBEC members use the same deamination activity to achieve diverse human biological functions. Deamination by an APOBEC protein called activation-induced cytidine deaminase (AID) is critical for generating high-affinity antibodies, and deamination by APOBEC-3 proteins can inhibit retrotransposons and the replication of retroviruses such as human immunodeficiency virus and hepatitis B virus. Here we report the crystal structure of APO2. APO2 forms a rod-shaped tetramer that differs markedly from the square-shaped tetramer of the free nucleotide cytidine deaminase, with which APOBEC proteins share considerable sequence homology. In APO2, two long alpha-helices of a monomer structure prevent the formation of a square-shaped tetramer and facilitate formation of the rod-shaped tetramer via head-to-head interactions of two APO2 dimers. Extensive sequence homology among APOBEC family members allows us to test APO2 structure-based predictions using AID. We show that AID deamination activity is impaired by mutations predicted to interfere with oligomerization and substrate access. The structure suggests how mutations in patients with hyper-IgM-2 syndrome inactivate AID, resulting in defective antibody maturation. 相似文献
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Many helicases modulate recombination, an essential process that needs to be tightly controlled. Mutations in some human disease helicases cause increased recombination, genome instability and cancer. To elucidate the potential mode of action of these enzymes, here we developed a single-molecule fluorescence assay that can visualize DNA binding and translocation of Escherichia coli Rep, a superfamily 1 DNA helicase homologous to Saccharomyces cerevisiae Srs2. Individual Rep monomers were observed to move on single-stranded (ss)DNA in the 3' to 5' direction using ATP hydrolysis. Strikingly, on hitting a blockade, such as duplex DNA or streptavidin, the protein abruptly snapped back close to its initial position, followed by further cycles of translocation and snapback. This repetitive shuttling is likely to be caused by a blockade-induced protein conformational change that enhances DNA affinity for the protein's secondary DNA binding site, thereby resulting in a transient DNA loop. Repetitive shuttling was also observed on ssDNA bounded by a stalled replication fork and an Okazaki fragment analogue, and the presence of Rep delayed formation of a filament of recombination protein RecA on ssDNA. Thus, the binding of a single Rep monomer to a stalled replication fork can lead to repetitive shuttling along the single-stranded region, possibly keeping the DNA clear of toxic recombination intermediates. 相似文献