<i>De novo</i>
            protein synthesis is required for activation-induced cytidine deaminase-dependent DNA cleavage in immunoglobulin class switch recombination

Begum, Noorzahan; Kinoshita, Kazuo; Muramatsu, Masamichi; Nagaoka, Hitoshi; Shinkura, Reiko; Honjo, Tasuku

doi:10.1073/pnas.0405219101

Cited by 38 publications

(24 citation statements)

References 36 publications

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“…We have shown that DSB associated with SHM depends on de novo protein synthesis but not on UNG. These features are the same as reported for CSR (24,25) and consistent with the RNA editing hypothesis but not with the DNA deamination hypothesis. Because AID has N-and C-terminal domains that are specifically required for SHM and CSR, respectively (10, 26), we proposed that AID interacts with SHM-and CSR-specific cofactors at the N and C termini, respectively.…”

Section: Aid-dependent ␥-H2ax Focus Formation On Ig Genes In Shm-indusupporting

confidence: 88%

“…ChIP was done as described (24,25). Nonspecific ␥-H2AX focus formation was induced by treatment of cells with 0.5 M staurosporine for 2 h as a positive control (30).…”

Section: Methodsmentioning

confidence: 99%

“…We showed that DSB in S regions depend on de novo protein synthesis but not on UNG (24,25). Furthermore, UNG mutants that abolished U removal activity still rescued class switching in UNG-deficient B cells (24).…”

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confidence: 99%

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DNA cleavage in immunoglobulin somatic hypermutation depends onde novoprotein synthesis but not on uracil DNA glycosylase

Nagaoka

Ito

Muramatsu

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Activation-induced cytidine deaminase (AID) is required for the DNA cleavage step of Ig somatic hypermutation (SHM). However, its molecular mechanism is controversial. The RNA editing hypothesis postulates that AID deaminates cytosine in an unknown mRNA to generate a new mRNA encoding SHM endonuclease. On the other hand, the DNA deamination hypothesis explains DNA cleavage by cytosine deamination in DNA, followed by uracil removal by uracil DNA glycosylase (UNG). By using the protein synthesis inhibitor cycloheximide, we showed that SHM requires de novo protein synthesis in accord with predictions by the RNA editing hypothesis. In addition, we found that cycloheximide but not Ugi (the specific inhibitor of UNG) inhibited AID-dependent DNA cleavage in the Ig gene during SHM, by using histone H2AX focus formation as a marker of DNA cleavage. The results indicate the following order of events: AID expression, protein synthesis, DNA cleavage, and SHM. The requirement of protein synthesis but not of UNG for the DNA cleavage step of SHM forces us to reconsider the DNA deamination hypothesis and strengthens the RNA editing hypothesis.A ntigen stimulation induces three types of genetic alterations, namely somatic hypermutation (SHM), gene conversion (GC), and class switch recombination (CSR) in activated B cells, giving rise to antigen-specific Ig with high affinity and appropriate effector functions. SHM introduces point mutations in variable-region (V) genes without template, whereas GC does so with pseudo-V genes as template. When SHM and GC are coupled with selection by limited amounts of antigen, highaffinity antibody-producing B cells are enriched. CSR is a genetic process that switches Ig isotypes from IgM to other isotypes such as IgG and IgE, adding diverse effector functions to Ig with a given antigen specificity (1, 2).Activation-induced cytidine deaminase (AID), which is strictly expressed in activated B cells, especially in the germinal centers of lymphoid follicles, is essential for all three types of DNA alteration in B cells activated by antigen stimulation (3-6). AID has the strongest homology with apolipoprotein B mRNA editing catalytic subunit 1 (APOBEC-1) (reviewed in ref. 7). The genetic loci for AID and APOBEC-1 are tightly linked on mouse and human chromosomes (2). In addition to these evolutionary conservations, AID has functional similarities with APOBEC-1, including dimer formation, requirement of cofactors, and shuttling between cytoplasm and nucleus by N-terminal nuclear localization and C-terminal nuclear export domains (8-10).Evolutionary conservation and biochemical similarities between AID and apolipoprotein B mRNA editing catalytic subunit 1 (APOBEC-1) led us to propose the RNA editing hypothesis that AID converts unknown mRNA precursors to novel mRNAs encoding putative endonucleases to cleave target DNA (1). The alternative hypothesis (DNA deamination) explains DNA cleavage by direct deamination of cytosine (C) to uracil (U) in target DNA by AID. To generate strand breakage, U should be...

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Section: Aid-dependent ␥-H2ax Focus Formation On Ig Genes In Shm-indusupporting

confidence: 88%

“…ChIP was done as described (24,25). Nonspecific ␥-H2AX focus formation was induced by treatment of cells with 0.5 M staurosporine for 2 h as a positive control (30).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

DNA cleavage in immunoglobulin somatic hypermutation depends onde novoprotein synthesis but not on uracil DNA glycosylase

Nagaoka

Ito

Muramatsu

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

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“…Although there are several hypotheses about how AID functions, data are accumulating that suggest that AID deaminates cytosine to uracil on the non-template strand of DNA during transcription (10). It is also possible that AID deaminates cytidine to uridine on an unknown mRNA (11,12). Data are still emerging that suggest AID has different roles and cofactor interactions when participating in CSR and SHM (13,14).…”

mentioning

confidence: 99%

Activation-induced deaminase cloning, localization, and protein extraction from young V_H-mutant rabbit appendix

Yang

Obiakor²,

Sinha³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Studies in mouse, human, and chicken suggest that activationinduced deaminase (AID) is involved in three known processes leading to antibody diversification: somatic hypermutation, gene conversion, and class-switch recombination. Developing rabbit appendix provides a particularly good site for studying all three of these B cell maturation events. We report here successful cloning of rabbit AID and isolation of AID protein from rabbit appendix-cell nuclear and cytoplasmic extracts. We succeeded in identifying and locating AID protein in cells by immunohistochemical and immunofluorescent staining techniques and examined colocalization of AID and other molecules important for Ab diversification. This report extends our knowledge about AID to a mammalian species that uses gene conversion to diversify rearranged Ig genes. Although much work remains to understand fully the mechanism of action of AID and its association with other cellular components, the rabbit system now offers a particularly useful model for future studies of these dynamics. mass spectrometry ͉ gene conversion ͉ somatic hypermutation T he rabbit is an excellent animal model for study of somatic hypermutation (SHM) and gene conversion (GC) of rearranged variable regions (V) of Ab heavy (H) and light (L) chain genes. The appendix of neonatal rabbits is a critical site for development of the primary Ab repertoire (1, 2). Rearrangements of H and L chain genes occur in sites including fetal liver, omentum, and bone marrow. After birth, B cells with rearranged genes migrate into gut-associated lymphoid tissue such as the appendix. Gut microflora are required for B cells to proliferate and develop in appendix follicles (3). At Ϸ3 wk of age, diversification of rearranged gene sequences by GC and SHM starts within the appendix-follicle microenvironment (2). GC diversifies sequences encoding V regions of H and L chains through introduction of sequences from upstream donor V gene segments. B cells that have undergone class-switch recombination (CSR), particularly to IgA, are also found in rabbit appendix at Ϸ3 wk of age (2). Activation-induced deaminase (AID) is involved in three processes of Ab diversification and maturation, SHM, CSR (4-7), and GC (8,9), that all coexist in young rabbit appendix. Thus far, the only published evidence for the role of AID in GC comes from two studies showing that in the chicken DT40 bursal tumor cell line that carries out GC in vitro, no GC occurs when AID is knocked out on both chromosomes (8, 9). Although there are several hypotheses about how AID functions, data are accumulating that suggest that AID deaminates cytosine to uracil on the non-template strand of DNA during transcription (10). It is also possible that AID deaminates cytidine to uridine on an unknown mRNA (11,12). Data are still emerging that suggest AID has different roles and cofactor interactions when participating in CSR and SHM (13,14).We used 1-to 6-wk-old ali͞ali V H mutant rabbits to study rabbit AID. These mutants have a small (Ϸ10 kb) deletion encompas...

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“…1b). The idea of an mRNA-editing function for AID is supported by the structural similarity of AID to APOBEC-1, an apolipoprotein B-editing enzyme, and the finding that de novo protein synthesis is required for CSR and for the formation of DNA repair γ-H2AX (phosphorylated H2AX, a mammalian core histone H2A variant) foci in the immunoglobulin heavy chain (Igh) locus 3 .…”

Section: Hhs Public Accessmentioning

confidence: 99%

Class switching and Myc translocation: how does DNA break?

Casali

Zan

2004

Nat Immunol

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Chromosomal translocations involving immunoglobulin switch regions are commonly thought to arise from aberrant AID-induced DNA lesions. New data, however, suggest AID does not initiate such lesions, but acts subsequently in the B cell transformation process.Early events in B cell malignant transformation often involve chromosomal translocations between the Myc oncogene and immunoglobulin switch (S) regions. These translocations are believed to arise from abberant immunoglobulin class-switch recombination (CSR), involving doublestrand DNA breaks (DSBs) in both Myc and the S regions. The enzyme activationinduced cytidine deaminase (AID) is required for CSR. DSBs in S regions are essential intermediates in CSR and are, together with DSBs in Myc, the initiating lesions leading to such translocations. Thus, one theory poses that AID is responsible for generating the aberrant lesions that initiate chromosomal translocations. However, new data from Schatz and colleagues 1 indicate that AID is not required for the first step of CSR: the generation of S-region DSBs. Unniraman et al. 1 show that this cytidine deaminase makes no measurable contribution to the generation of initial Myc-S-region translocations, thereby indicating that S regions accumulate DSBs in a way that does not require the intervention of AID.The genes encoding the B cell receptor for antigen and secreted antibody show a high degree of variability, which is generated by two sequential stages of genetic alteration. In the early stage, gene recombination dependent on the recombination activating gene product selects one of each of the variable (V), diversity (D) and joining (J) segments from the respective gene pools and combines them into a single immunoglobulin V(D)J exon. After a B cell encounters its corresponding antigen, immunoglobulin genes undergo two types of DNA modification: CSR and somatic hypermutation (SHM). CSR and SHM somatically diversify the antibody molecule in different ways, although both require AID. CSR substitutes the constant region of the antibody heavy chain (C H ) with a downstream C H region by introducing DSBs in the S regions of an upstream and a downstream C H gene and perfecting an intrachromosomal and inter-S-S-region recombination entailing the excision of intervening genomic DNA. SHM inserts single-nucleotide changes (mismatches) in V(D)J genes.The demonstration that AID can directly deaminate DNA in Escherichia coli 2 and is upregulated in germinal center B cells, which actively undergo CSR and can incur into neoplastic Myc translocation, has led to the assumption that AID effects DSBs in the S

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De novo protein synthesis is required for activation-induced cytidine deaminase-dependent DNA cleavage in immunoglobulin class switch recombination

Cited by 38 publications

References 36 publications

DNA cleavage in immunoglobulin somatic hypermutation depends onde novoprotein synthesis but not on uracil DNA glycosylase

DNA cleavage in immunoglobulin somatic hypermutation depends onde novoprotein synthesis but not on uracil DNA glycosylase

Activation-induced deaminase cloning, localization, and protein extraction from young V_H-mutant rabbit appendix

Class switching and Myc translocation: how does DNA break?

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De novo protein synthesis is required for activation-induced cytidine deaminase-dependent DNA cleavage in immunoglobulin class switch recombination

Cited by 38 publications

References 36 publications

DNA cleavage in immunoglobulin somatic hypermutation depends onde novoprotein synthesis but not on uracil DNA glycosylase

DNA cleavage in immunoglobulin somatic hypermutation depends onde novoprotein synthesis but not on uracil DNA glycosylase

Activation-induced deaminase cloning, localization, and protein extraction from young VH-mutant rabbit appendix

Class switching and Myc translocation: how does DNA break?

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Activation-induced deaminase cloning, localization, and protein extraction from young V_H-mutant rabbit appendix