scholarly journals DNA-PKcs and Artemis function in the end-joining phase of immunoglobulin heavy chain class switch recombination

2008 ◽  
Vol 205 (3) ◽  
pp. 557-564 ◽  
Author(s):  
Sonia Franco ◽  
Michael M. Murphy ◽  
Gang Li ◽  
Tiffany Borjeson ◽  
Cristian Boboila ◽  
...  
Keyword(s):  
B Cells ◽  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Class Switch ◽  
Chromosomal Breaks ◽  
Dependent Protein ◽  
Activation Conditions

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and Artemis are classical nonhomologous DNA end-joining (C-NHEJ) factors required for joining a subset of DNA double-strand breaks (DSB), particularly those requiring end processing. In mature B cells, activation-induced cytidine deaminase (AID) initiates class switch recombination (CSR) by introducing lesions into S regions upstream of two recombining CH exons, which are processed into DSBs and rejoined by C-NHEJ to complete CSR. The function of DNA-PKcs in CSR has been controversial with some reports but not others showing that DNA-PKcs–deficient mice are significantly impaired for CSR. Artemis-deficient B cells reportedly undergo CSR at normal levels. Overall, it is still not known whether there are any CSR-associated DSBs that require DNA-PKcs and/or Artemis to be joined. Here, we have used an immunoglobulin (Ig)H locus-specific fluorescent in situ hybridization assay to unequivocally demonstrate that both DNA-PKcs and, unexpectedly, Artemis are necessary for joining a subset of AID-dependent DSBs. In the absence of either factor, B cells activated for CSR frequently generate AID-dependent IgH locus chromosomal breaks and translocations. We also find that under specific activation conditions, DNA-PKcs−/− B cells with chromosomal breaks are eliminated or at least prevented from progressing to metaphase via a p53-dependent response.

scholarly journals BRCT-domain protein BRIT1 influences class switch recombination

2017 ◽  
Vol 114 (31) ◽  
pp. 8354-8359 ◽  
Author(s):  
Wei-Feng Yen ◽  
Ashutosh Chaudhry ◽  
Bharat Vaidyanathan ◽  
William T. Yewdell ◽  
Joseph N. Pucella ◽  
...  
Keyword(s):  
B Cells ◽  
Ex Vivo ◽  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
B Cell Lymphoma ◽  
Brct Domain ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Class Switch ◽  
Domain Protein

DNA double-strand breaks (DSBs) serve as obligatory intermediates for Ig heavy chain (Igh) class switch recombination (CSR). The mechanisms by which DSBs are resolved to promote long-range DNA end-joining while suppressing genomic instability inherently associated with DSBs are yet to be fully elucidated. Here, we use a targeted short-hairpin RNA screen in a B-cell lymphoma line to identify the BRCT-domain protein BRIT1 as an effector of CSR. We show that conditional genetic deletion of BRIT1 in mice leads to a marked increase in unrepaired Igh breaks and a significant reduction in CSR in ex vivo activated splenic B cells. We find that the C-terminal tandem BRCT domains of BRIT1 facilitate its interaction with phosphorylated H2AX and that BRIT1 is recruited to the Igh locus in an activation-induced cytidine deaminase (AID) and H2AX-dependent fashion. Finally, we demonstrate that depletion of another BRCT-domain protein, MDC1, in BRIT1-deleted B cells increases the severity of CSR defect over what is observed upon loss of either protein alone. Our results identify BRIT1 as a factor in CSR and demonstrate that multiple BRCT-domain proteins contribute to optimal resolution of AID-induced DSBs.

scholarly journals Histone methyltransferase MMSET promotes AID-mediated DNA breaks at the donor switch region during class switch recombination

2017 ◽  
Vol 114 (49) ◽  
pp. E10560-E10567 ◽  
Author(s):  
Hai Vu Nguyen ◽  
Junchao Dong ◽  
Rohit A. Panchakshari ◽  
Vipul Kumar ◽  
Frederick W. Alt ◽  
...  
Keyword(s):  
B Cells ◽  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
Histone Methyltransferase ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Set Domain ◽  
End Joining ◽  
Class Switch

In B cells, Ig class switch recombination (CSR) is initiated by activation-induced cytidine deaminase (AID), the activity of which leads to DNA double-strand breaks (DSBs) within IgH switch (S) regions. Preferential targeting of AID-mediated DSBs to S sequences is critical for allowing diversification of antibody functions, while minimizing potential off-target oncogenic events. Here, we used gene targeted inactivation of histone methyltransferase (HMT) multiple myeloma SET domain (MMSET) in mouse B cells and the CH12F3 cell line to explore its role in CSR. We find that deletion of MMSET-II, the isoform containing the catalytic SET domain, inhibits CSR without affecting either IgH germline transcription or joining of DSBs within S regions by classical nonhomologous end joining (C-NHEJ). Instead, we find that MMSET-II inactivation leads to decreased AID recruitment and DSBs at the upstream donor Sμ region. Our findings suggest a role for the HMT MMSET in promoting AID-mediated DNA breaks during CSR.

scholarly journals Involvement of Artemis in nonhomologous end-joining during immunoglobulin class switch recombination

2008 ◽  
Vol 205 (13) ◽  
pp. 3031-3040 ◽  
Author(s):  
Likun Du ◽  
Mirjam van der Burg ◽  
Sergey W. Popov ◽  
Ashwin Kotnis ◽  
Jacques J.M. van Dongen ◽  
...  
Keyword(s):  
Class Switch Recombination ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Igg Subclass ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Immunoglobulin Class ◽  
Class Switch ◽  
Human B Cells

DNA double-strand breaks (DSBs) introduced in the switch (S) regions are intermediates during immunoglobulin class switch recombination (CSR). These breaks are subsequently recognized, processed, and joined, leading to recombination of the two S regions. Nonhomologous end-joining (NHEJ) is believed to be the principle mechanism involved in DSB repair during CSR. One important component in NHEJ, Artemis, has however been considered to be dispensable for efficient CSR. In this study, we have characterized the S recombinational junctions from Artemis-deficient human B cells. Sμ–Sα junctions could be amplified from all patients tested and were characterized by a complete lack of “direct” end-joining and a remarkable shift in the use of an alternative, microhomology-based end-joining pathway. Sμ–Sγ junctions could only be amplified from one patient who carries “hypomorphic” mutations. Although these Sμ–Sγ junctions appear to be normal, a significant increase of an unusual type of sequential switching from immunoglobulin (Ig)M, through one IgG subclass, to a different IgG subclass was observed, and the Sγ–Sγ junctions showed long microhomologies. Thus, when the function of Artemis is impaired, varying modes of CSR junction resolution may be used for different S regions. Our findings strongly link Artemis to the predominant NHEJ pathway during CSR.

scholarly journals Kinase-dependent structural role of DNA-PKcs during immunoglobulin class switch recombination

2018 ◽  
Vol 115 (34) ◽  
pp. 8615-8620 ◽  
Author(s):  
Jennifer L. Crowe ◽  
Zhengping Shao ◽  
Xiaobin S. Wang ◽  
Pei-Chi Wei ◽  
Wenxia Jiang ◽  
...  
Keyword(s):  
B Cells ◽  
High Throughput Sequencing ◽  
Class Switch Recombination ◽  
End Joining ◽  
Extensive Resection ◽  
Structural Role ◽  
Class Switch ◽  
Cell Class ◽  
Dependent Protein

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is a classical nonhomologous end-joining (cNHEJ) factor. Loss of DNA-PKcs diminished mature B cell class switch recombination (CSR) to other isotypes, but not IgG1. Here, we show that expression of the kinase-dead DNA-PKcs (DNA-PKcsKD/KD) severely compromises CSR to IgG1. High-throughput sequencing analyses of CSR junctions reveal frequent accumulation of nonproductive interchromosomal translocations, inversions, and extensive end resection in DNA-PKcsKD/KD, but not DNA-PKcs−/−, B cells. Meanwhile, the residual joints from DNA-PKcsKD/KD cells and the efficient Sµ-Sγ1 junctions from DNA-PKcs−/− B cells both display similar preferences for small (2–6 nt) microhomologies (MH). In DNA-PKcs−/− cells, Sµ-Sγ1 joints are more resistant to inversions and extensive resection than Sµ-Sε and Sµ-Sµ joints, providing a mechanism for the isotype-specific CSR defects. Together, our findings identify a kinase-dependent role of DNA-PKcs in suppressing MH-mediated end joining and a structural role of DNA-PKcs protein in the orientation of CSR.

scholarly journals 53BP1 regulates DNA resection and the choice between classical and alternative end joining during class switch recombination

2010 ◽  
Vol 207 (4) ◽  
pp. 855-865 ◽  
Author(s):  
Anne Bothmer ◽  
Davide F. Robbiani ◽  
Niklas Feldhahn ◽  
Anna Gazumyan ◽  
Andre Nussenzweig ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Response Factors ◽  
Class Switch ◽  
Damage Response ◽  
Switch Regions

Class switch recombination (CSR) diversifies antibodies by joining highly repetitive DNA elements, which are separated by 60–200 kbp. CSR is initiated by activation-induced cytidine deaminase, an enzyme that produces multiple DNA double-strand breaks (DSBs) in switch regions. Switch regions are joined by a mechanism that requires an intact DNA damage response and classical or alternative nonhomologous end joining (A-NHEJ). Among the DNA damage response factors, 53BP1 has the most profound effect on CSR. We explore the role of 53BP1 in intrachromosomal DNA repair using I-SceI to introduce paired DSBs in the IgH locus. We find that the absence of 53BP1 results in an ataxia telangiectasia mutated–dependent increase in DNA end resection and that resected DNA is preferentially repaired by microhomology-mediated A-NHEJ. We propose that 53BP1 favors long-range CSR in part by protecting DNA ends against resection, which prevents A-NHEJ–dependent short-range rejoining of intra–switch region DSBs.

scholarly journals MBD4 Facilitates Immunoglobulin Class Switch Recombination

2016 ◽  
Vol 37 (2) ◽  
Author(s):  
Fernando Grigera ◽  
Robert Wuerffel ◽  
Amy L. Kenter
Keyword(s):  
Excision Repair ◽  
Cytidine Deaminase ◽  
Ectopic Expression ◽  
Class Switch Recombination ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Class Switch ◽  
Activation Induced Cytidine Deaminase ◽  
Base Excision

ABSTRACT Immunoglobulin heavy chain class switch recombination (CSR) requires targeted formation of DNA double-strand breaks (DSBs) in repetitive switch region elements followed by ligation between distal breaks. The introduction of DSBs is initiated by activation-induced cytidine deaminase (AID) and requires base excision repair (BER) and mismatch repair (MMR). The BER enzyme methyl-CpG binding domain protein 4 (MBD4) has been linked to the MMR pathway through its interaction with MutL homologue 1 (MLH1). We find that when Mbd4 exons 6 to 8 are deleted in a switching B cell line, DSB formation is severely reduced and CSR frequency is impaired. Impaired CSR can be rescued by ectopic expression of Mbd4. Mbd4 deficiency yields a deficit in DNA end processing similar to that found in MutS homologue 2 (Msh2)- and Mlh1-deficient B cells. We demonstrate that microhomology-rich S-S junctions are enriched in cells in which Mbd4 is deleted. Our studies suggest that Mbd4 is a component of MMR-directed DNA end processing.

scholarly journals Non-homologous end joining in class switch recombination: the beginning of the end

2008 ◽  
Vol 364 (1517) ◽  
pp. 653-665 ◽  
Author(s):  
Ashwin Kotnis ◽  
Likun Du ◽  
Chonghai Liu ◽  
Sergey W Popov ◽  
Qiang Pan-Hammarström
Keyword(s):  
Ataxia Telangiectasia ◽  
Current Knowledge ◽  
Class Switch Recombination ◽  
Specific Factor ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Class Switch ◽  
Dependent Protein ◽  
Activation Induced Deaminase ◽  
Non Homologous End Joining

Immunoglobulin class switch recombination (CSR) is initiated by a B-cell-specific factor, activation-induced deaminase, probably through deamination of deoxycytidine residues within the switch (S) regions. The initial lesions in the S regions are subsequently processed, resulting in the production of DNA double-strand breaks (DSBs). These breaks will then be recognized, edited and repaired, finally leading to the recombination of the two S regions. Two major repair pathways have been implicated in CSR, the predominant non-homologous end joining (NHEJ) and the alternative end-joining (A-EJ) pathways. The former requires not only components of the ‘classical’ NHEJ machinery, i.e. Ku70/Ku80, DNA-dependent protein kinase catalytic subunit, DNA ligase IV and XRCC4, but also a number of DNA-damage sensors or adaptors, such as ataxia–telangiectasia mutated, γH2AX, 53BP1, MDC1, the Mre11–Rad50–NBS1 complex and the ataxia telangiectasia and Rad3-related protein (ATR). The latter pathway is not well characterized yet and probably requires microhomologies. In this review, we will focus on the current knowledge of the predominant NHEJ pathway in CSR and will also give a perspective on the A-EJ pathway.

scholarly journals Altered kinetics of nonhomologous end joining and class switch recombination in ligase IV–deficient B cells

2008 ◽  
Vol 205 (12) ◽  
pp. 2745-2753 ◽  
Author(s):  
Li Han ◽  
Kefei Yu
Keyword(s):  
Class Switch Recombination ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Class Switch ◽  
Ligase Iv ◽  
Switch Regions ◽  
Cytokine Stimulation

Immunoglobulin heavy chain class switch recombination (CSR) is believed to occur through the generation and repair of DNA double-strand breaks (DSBs) in the long and repetitive switch regions. Although implied, the role of the major vertebrate DSB repair pathway, nonhomologous end joining (NHEJ), in CSR has been controversial. By somatic gene targeting of DNA ligase IV (Lig4; a key component of NHEJ) in a B cell line (CH12F3) capable of highly efficient CSR in vitro, we found that NHEJ is required for efficient CSR. Disruption of the Lig4 gene in CH12F3 cells severely inhibits the initial rate of CSR and causes a late cell proliferation defect under cytokine stimulation. However, unlike V(D)J recombination, which absolutely requires NHEJ, CSR accumulates to a substantial level in Lig4-null cells. The data revealed a fast-acting NHEJ and a slow-acting alterative end joining of switch region breaks during CSR.

scholarly journals Oncogenic transformation in the absence of Xrcc4 targets peripheral B cells that have undergone editing and switching

2008 ◽  
Vol 205 (13) ◽  
pp. 3079-3090 ◽  
Author(s):  
Jing H. Wang ◽  
Frederick W. Alt ◽  
Monica Gostissa ◽  
Abhishek Datta ◽  
Michael Murphy ◽  
...  
Keyword(s):  
B Cells ◽  
Chromosomal Translocation ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Class Switch ◽  
Ig Heavy Chain ◽  
Peripheral B Cells

Nonhomologous end-joining (NHEJ) repairs DNA double-strand breaks (DSBs) during V(D)J recombination in developing lymphocytes and during immunoglobulin (Ig) heavy chain (IgH) class switch recombination (CSR) in peripheral B lymphocytes. We now show that CD21-cre–mediated deletion of the Xrcc4 NHEJ gene in p53-deficient peripheral B cells leads to recurrent surface Ig-negative B lymphomas (“CXP lymphomas”). Remarkably, CXP lymphomas arise from peripheral B cells that had attempted both receptor editing (secondary V[D]J recombination of Igκ and Igλ light chain genes) and IgH CSR subsequent to Xrcc4 deletion. Correspondingly, CXP tumors frequently harbored a CSR-based reciprocal chromosomal translocation that fused IgH to c-myc, as well as large chromosomal deletions or translocations involving Igκ or Igλ, with the latter fusing Igλ to oncogenes or to IgH. Our findings reveal peripheral B cells that have undergone both editing and CSR and show them to be common progenitors of CXP tumors. Our studies also reveal developmental stage-specific mechanisms of c-myc activation via IgH locus translocations. Thus, Xrcc4/p53-deficient pro–B lymphomas routinely activate c-myc by gene amplification, whereas Xrcc4/p53-deficient peripheral B cell lymphomas routinely ectopically activate a single c-myc copy.

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