The Sμ Tandem Repeat Region Is Critical for Ig Isotype Switching in the Absence of Msh2

Min, Irene M.; Schrader, Carol E.; Vardo, Joycelyn; Luby, Thomas M.; D'Avirro, Nicole; Stavnezer, Janet; Selsing, Erik

doi:10.1016/s1074-7613(03)00262-0

Cited by 58 publications

(74 citation statements)

References 51 publications

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“…In fact, the level of residual switching observed in APE1-deficient cells in this study is remarkably similar to that observed in cells deficient in UNG2. MMR has been shown to be particularly important when core switch repeats were deleted and overlapping AID hot spots were scarce (10,32,33). Among the MMR factors, the MutL␣ (MLH1/ PMS2) complex has been reported to possess a latent endonuclease activity that is activated upon mismatch recognition (34).…”

Section: Discussionmentioning

confidence: 99%

Apurinic/Apyrimidinic Endonuclease 1 Is the Essential Nuclease during Immunoglobulin Class Switch Recombination

Masani

Han

2013

Molecular and Cellular Biology

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Immunoglobulin (Ig) class switch recombination (CSR) is initiated by activation-induced cytidine deaminase (AID) that catalyzes numerous DNA cytosine deaminations within switch regions. The resulting uracils are processed by uracil base excision and/or mismatch repair enzymes that ultimately generate switch region DNA double-strand breaks (DSBs). Uracil glycosylase 2 (UNG2) is required for CSR, most likely by removing uracils to generate abasic sites. Although it is presumed that the apurinic/ apyrimidinic endonuclease 1 (APE1) generates DNA strand incisions (a prerequisite for CSR) at these abasic sites, a direct test of the requirement for APE1 in CSR has been difficult because of the embryonic lethality of APE1 ablation in mice. Here, we report the successful deletion of the APE1 gene in a mouse B cell line (CH12F3) capable of robust CSR in vitro. In contrast to the general assumption that APE1 is essential for cellular viability, deletion of APE1 in CH12F3 cells has no apparent effect on cell viability or growth. Moreover, CSR in APE1-null CH12F3 cells is drastically reduced, providing direct evidence for an essential role for APE1 in switch region cleavage and CSR. Finally, deletion of AP endonuclease 2 (APE2) has no effect on CSR in either APE1-proficient or -deficient cells. In antigen-stimulated B cells, somatic hypermutation (SHM) diversifies the variable regions of immunoglobulin (Ig) heavy (H) and light (L) chain genes, whereas class switch recombination (CSR) provides a mechanism to diversify the constant region of the Ig heavy chain (1, 2). SHM introduces point mutations into the Ig variable (V) regions, which is the mechanistic basis for Ig affinity maturation. CSR introduces DNA double-strand breaks (DSBs) into donor and acceptor switch (S) regions; subsequently a "cut-and-paste" mechanism results in intrachromosomal deletion. Thus, CSR juxtaposes a previously distal constant region to the V region, allowing a "switch" of the class (or isotype) of the expressed immunoglobulin without altering its antigen specificity (1, 2).Both SHM and CSR require activation-induced cytidine deaminase (AID) (3, 4), which deaminates DNA cytosines to uracils at transcribed V and S regions (1, 2, 5). Uracils in DNA can be detected and repaired by either the uracil glycosylase (UNG2)-dependent base excision repair (BER) pathway or by the MSH2-dependent mismatch repair (MMR) pathway. Indeed, deletion of either UNG2 or MSH2 reduces CSR efficiency (6, 7) and deletion of both completely abolishes CSR (8). However, in contrast to the normal high-fidelity repair that restores DNA to its original state, processing of AID-generated uracils leads to mutations in the V region during SHM and DNA double-strand breaks in switch regions during CSR. It is therefore unclear whether the BER and MMR factors that are involved in SHM and CSR have been usurped to function differently (without their characteristic high fidelity) in germinal center B cells or whether perhaps only some of the components in either pathway are utilized.A notic...

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Section: Discussionmentioning

confidence: 99%

Apurinic/Apyrimidinic Endonuclease 1 Is the Essential Nuclease during Immunoglobulin Class Switch Recombination

Masani

Han

2013

Molecular and Cellular Biology

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“…In contrast, SSBs in opposite strands resulting from deamination of two relatively distant dC need processing by the MMR pathway to be converted into DSBs, which might only then trigger the accumulation of gH2AX and 53BP1 at the damage site (44,45,52). Accordingly, MMR is particularly important to allow CSR using the pre-Sm when the Sm tandem repeats are deleted (53), because the lower AID hot spot frequency in the pre-Sm will most preferentially accommodate staggered breaks. The proposed model for processing SSBs by MMR during CSR predicts that the U:G mismatch is recognized by the MSH2/MSH6 complex.…”

Section: Discussionmentioning

confidence: 99%

Alternative End-Joining and Classical Nonhomologous End-Joining Pathways Repair Different Types of Double-Strand Breaks during Class-Switch Recombination

Cortizas

Zahn

Hajjar

et al. 2013

The Journal of Immunology

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Classical nonhomologous end-joining (C-NHEJ) and alternative end-joining (A-EJ) are the main DNA double-strand break (DSB) repair pathways when a sister chromatid is not available. However, it is not clear how one pathway is chosen over the other to process a given DSB. To address this question, we studied in mouse splenic B cells and CH12F3 cells how C-NHEJ and A-EJ repair DSBs initiated by the activation-induced deaminase during IgH (Igh) class-switch recombination (CSR). We show in this study that lowering the deamination density at the Igh locus increases DSB resolution by microhomology-mediated repair while decreasing C-NHEJ activity. This process occurs without affecting 53BP1 and γH2AX levels during CSR. Mechanistically, lowering deamination density increases exonuclease I recruitment and single-stranded DNA at the Igh locus and promotes C-terminal binding protein interacting protein and MSH2-dependent DSB repair during CSR. Indeed, reducing activation-induced deaminase levels increases CSR efficiency in C-NHEJ–defective cells, suggesting enhanced use of an A-EJ pathway. Our results establish a mechanism by which C-NHEJ and this C-terminal binding protein interacting protein/MSH2-dependent pathway that relies on microhomology can act concurrently but independently to repair different types of DSBs and reveal that the density of DNA lesions influences the choice of DSB repair pathway during CSR.

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“…3), some level of residual CSR occurs upstream and downstream of the position of the deletion boundary (20,21), and the ones occurring upstream in the core S deletion mice would be particularly difficult to explain if R-loops initiated only in the core S region. Because we now know that the R-loops can begin upstream, the breakpoints upstream of the core S in the deletion mice can now be more easily understood.…”

Section: Discussionmentioning

confidence: 99%

Sequence Dependence of Chromosomal R-Loops at the Immunoglobulin Heavy-Chain Sμ Class Switch Region

Huang¹,

Yu²,

Balter

et al. 2007

Molecular and Cellular Biology

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101

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The mechanism by which the cytidine deaminase activation-induced deaminase (AID) acts at immunoglobulin heavy-chain class switch regions during mammalian class switch recombination (CSR) remains unclear. R-loops have been proposed as a basis for this targeting. Here, we show that the difference between various forms of the S locus that can or cannot undergo CSR correlates well with the locations and detectability of R-loops. The S R-loops can initiate hundreds of base pairs upstream of the core repeat switch regions, and the area where the R-loops initiate corresponds to the zone where the AID mutation frequency begins to rise, despite a constant density of WRC sites in this region. The frequency of R-loops is 1 in 25 alleles, regardless of the presence of the core S repeats, again consistent with the initiation of most R-loops upstream of the core repeats. These findings explain the surprisingly high levels of residual CSR in B cells from mice lacking the core S repeats but the marked reduction in CSR in mice with deletions of the region upstream of the core S repeats. These studies also provide the first analysis of how R-loop formation in the eukaryotic chromosome depends on the DNA sequence.Mammalian immunoglobulin (Ig) genes undergo two types of DNA recombination, in addition to a somatic hypermutation (SHM) process. V(D)J recombination occurs in early lymphocytes and assembles the variable-domain exon so that IgM can be made. Class switch recombination (CSR) occurs only at the Ig heavy-chain locus and is responsible for the change in the heavy-chain isotype from IgM to IgG, IgA, or IgE; this process is also called the heavy-chain isotype switch (6,17,29). CSR occurs at repetitive DNA elements called switch regions, which vary in sequence and length. All of the switch regions are more than 1 kb in length and consist of unit repeats of 25 to 80 bp. All are located downstream of a sterile transcript promoter, which is necessary for CSR. All have a G-rich nontemplate strand, and all are rich in sites at which a cytidine deaminase called activation-induced deaminase (AID) prefers to act, namely, WRC sites (37). The regional nature of CSR, which gave rise to the term regionally specific recombination (15), contrasts with the vast majority of other physiologic recombination systems in biology, which are regarded as site specific. The special features of switch regions (such as being long and repetitive and located downstream of a promoter, having Grich nontemplate strands, and not having sequences conserved among the different switch regions or among vertebrates that carry out CSR) suggested that the mechanism would be unusual relative to other recombination processes in biology.Investigators at the Honjo laboratory discovered the key lymphoid-specific enzyme for both CSR and SHM, AID (22,23). AID is a 26-kDa protein which deaminates C in DNA (5, 25) but only when that DNA is single stranded (2,26,36,38). A key question for CSR and SHM concerns how the DNA becomes single stranded. Because transcription appears to...

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The Sμ Tandem Repeat Region Is Critical for Ig Isotype Switching in the Absence of Msh2

Cited by 58 publications

References 51 publications

Apurinic/Apyrimidinic Endonuclease 1 Is the Essential Nuclease during Immunoglobulin Class Switch Recombination

Apurinic/Apyrimidinic Endonuclease 1 Is the Essential Nuclease during Immunoglobulin Class Switch Recombination

Alternative End-Joining and Classical Nonhomologous End-Joining Pathways Repair Different Types of Double-Strand Breaks during Class-Switch Recombination

Sequence Dependence of Chromosomal R-Loops at the Immunoglobulin Heavy-Chain Sμ Class Switch Region

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