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 Parp1 facilitates alternative NHEJ, whereas Parp2 suppresses IgH/c-myc translocations during immunoglobulin class switch recombination

2009 ◽  
Vol 206 (5) ◽  
pp. 1047-1056 ◽  
Author(s):  
Isabelle Robert ◽  
Françoise Dantzer ◽  
Bernardo Reina-San-Martin
Keyword(s):  
Dna Damage ◽  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
Dependent Manner ◽  
Dna Breaks ◽  
End Joining ◽  
Immunoglobulin Class ◽  
Class Switch ◽  
Switch Regions ◽  
Alternative Nhej

Immunoglobulin class switch recombination (CSR) is initiated by DNA breaks triggered by activation-induced cytidine deaminase (AID). These breaks activate DNA damage response proteins to promote appropriate repair and long-range recombination. Aberrant processing of these breaks, however, results in decreased CSR and/or increased frequency of illegitimate recombination between the immunoglobulin heavy chain locus and oncogenes like c-myc. Here, we have examined the contribution of the DNA damage sensors Parp1 and Parp2 in the resolution of AID-induced DNA breaks during CSR. We find that although Parp enzymatic activity is induced in an AID-dependent manner during CSR, neither Parp1 nor Parp2 are required for CSR. We find however, that Parp1 favors repair of switch regions through a microhomology-mediated pathway and that Parp2 actively suppresses IgH/c-myc translocations. Thus, we define Parp1 as facilitating alternative end-joining and Parp2 as a novel translocation suppressor during CSR.

scholarly journals The cohesin complex regulates immunoglobulin class switch recombination

2013 ◽  
Vol 210 (12) ◽  
pp. 2495-2502 ◽  
Author(s):  
Anne-Sophie Thomas-Claudepierre ◽  
Ebe Schiavo ◽  
Vincent Heyer ◽  
Marjorie Fournier ◽  
Adeline Page ◽  
...  
Keyword(s):  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
Nonhomologous End Joining ◽  
Cohesin Complex ◽  
Dna Breaks ◽  
End Joining ◽  
Regulatory Subunits ◽  
Class Switch ◽  
Chromatid Cohesion ◽  
Switch Regions

Immunoglobulin (Ig) class switch recombination (CSR) is initiated by the transcription-coupled recruitment of activation-induced cytidine deaminase (AID) to switch regions and by the subsequent generation of double-stranded DNA breaks (DSBs). These DNA breaks are ultimately resolved through the nonhomologous end joining (NHEJ) pathway. We show that during CSR, AID associates with subunits of cohesin, a complex previously implicated in sister chromatid cohesion, DNA repair, and the formation of DNA loops between enhancers and promoters. Furthermore, we implicate the cohesin complex in the mechanism of CSR by showing that cohesin is dynamically recruited to the Sμ-Cμ region of the IgH locus during CSR and that knockdown of cohesin or its regulatory subunits results in impaired CSR and increased usage of microhomology-based end joining.

53BP1 links DNA damage-response pathways to immunoglobulin heavy chain class-switch recombination

2004 ◽  
Vol 5 (5) ◽  
pp. 481-487 ◽  
Author(s):  
John P Manis ◽  
Julio C Morales ◽  
Zhenfang Xia ◽  
Jeffery L Kutok ◽  
Frederick W Alt ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Heavy Chain ◽  
Class Switch Recombination ◽  
Immunoglobulin Heavy Chain ◽  
Class Switch ◽  
Damage Response

scholarly journals 53BP1 deficiency combined with telomere dysfunction activates ATR-dependent DNA damage response

2012 ◽  
Vol 197 (2) ◽  
pp. 283-300 ◽  
Author(s):  
Paula Martínez ◽  
Juana M. Flores ◽  
Maria A. Blasco
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Ataxia Telangiectasia ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Damage Response

TRF1 protects mammalian telomeres from fusion and fragility. Depletion of TRF1 leads to telomere fusions as well as accumulation of γ-H2AX foci and activation of both the ataxia telangiectasia mutated (ATM)– and the ataxia telangiectasia and Rad3 related (ATR)–mediated deoxyribonucleic acid (DNA) damage response (DDR) pathways. 53BP1, which is also present at dysfunctional telomeres, is a target of ATM that accumulates at DNA double-strand breaks and favors nonhomologous end-joining (NHEJ) repair over ATM-dependent resection and homology-directed repair (homologous recombination [HR]). To address the role of 53BP1 at dysfunctional telomeres, we generated mice lacking TRF1 and 53BP1. 53BP1 deficiency significantly rescued telomere fusions in mouse embryonic fibroblasts (MEFs) lacking TRF1, but they showed evidence of a switch from the NHEJ- to HR-mediated repair of uncapped telomeres. Concomitantly, double-mutant MEFs showed evidence of hyperactivation of the ATR-dependent DDR. In intact mice, combined 53BP1/TRF1 deficiency in stratified epithelia resulted in earlier onset of DNA damage and increased CHK1 phosphorylation during embryonic development, leading to aggravation of skin phenotypes.

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.

53BP1 Is Targeted to Sites of DNA Breaks within the Immunoglobulin Heavy Chain Locus and Promotes Class Switch Recombination.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2379-2379
Author(s):  
John Manis ◽  
Nicole Walsh ◽  
Phil Carpenter ◽  
Shilpee Dutt
Keyword(s):  
Dna Damage ◽  
B Cells ◽  
Dna Damage Response ◽  
Cellular Response ◽  
Somatic Hypermutation ◽  
Class Switch Recombination ◽  
Dna Breaks ◽  
Class Switch ◽  
Damage Response

Abstract The maintenance of genomic integrity relies on the cellular response to chromosomal damage from both exogenous (e.g. ionizing radiation) and endogenous (e.g. oxidative stress) sources. Various members of the DNA damage-sensing pathway including ATM, H2AX, 53BP1, and MDC1 are necessary to orchestrate the repair of DNA breaks. B cells undergo several programmed DNA alterations during their development: V(D)J recombination, Somatic Hypermutation (SHM), and Class Switch Recombination (CSR). We have previously shown that 53BP1 is relatively dispensable for V(D)J recombination and SHM. In contrast, class switch recombination is largely blocked to all isotypes indicating that regulated DNA breaks in B cells are regarded differentially by the DNA damage response machinery. 53BP1 is thought to promote the joining of DNA ends during CSR thus preventing translocations that could potentially lead to lymphoma. To better understand the damage response to CSR induced DNA breaks, a chromatin immunoprecipitation strategy and a combined immunofluorescence/FISH method was used to examine the components that assemble at IgH switch (S) regions during CSR. H2AX was found at S regions specifically targeted to undergo CSR after in vitro stimulation of B cells, and to a lesser degree, at adjacent S regions that were not activated for a switch event. H2AX was also found at S regions in switch activated 53BP1-deficient B cells. In contrast, 53BP1 was found primarily at S regions specifically targeted for CSR, and not at the adjacent S regions. Moreover, the localization of 53BP1 to S regions appeared to be in part, independent of DNA breaks, and potentially reliant on specialized DNA structures that are generated during CSR. These findings support a differential role for the various components of the DNA damage response program during CSR and have implications for understanding mechanisms of lymphomagenesis.

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 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 DNA-PKcs phosphorylation at the T2609 cluster alters the repair pathway choice during immunoglobulin class switch recombination

2020 ◽  
Vol 117 (37) ◽  
pp. 22953-22961 ◽  
Author(s):  
Jennifer L. Crowe ◽  
Xiaobin S. Wang ◽  
Zhengping Shao ◽  
Brian J. Lee ◽  
Verna M. Estes ◽  
...  
Keyword(s):  
Dna Repair ◽  
B Cells ◽  
Class Switch Recombination ◽  
Radiation Sensitivity ◽  
Repair Pathway ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Class Switch ◽  
Switch Regions ◽  
Pathway Choice

The DNA-dependent protein kinase (DNA-PK), which is composed of the KU heterodimer and the large catalytic subunit (DNA-PKcs), is a classical nonhomologous end-joining (cNHEJ) factor. Naïve B cells undergo class switch recombination (CSR) to generate antibodies with different isotypes by joining two DNA double-strand breaks at different switching regions via the cNHEJ pathway. DNA-PK and the cNHEJ pathway play important roles in the DNA repair phase of CSR. To initiate cNHEJ, KU binds to DNA ends and recruits and activates DNA-PK. Activated DNA-PK phosphorylates DNA-PKcs at the S2056 and T2609 clusters. Loss of T2609 cluster phosphorylation increases radiation sensitivity but whether T2609 phosphorylation has a role in physiological DNA repair remains elusive. Using the DNA-PKcs5A mouse model carrying alanine substitutions at the T2609 cluster, here we show that loss of T2609 phosphorylation of DNA-PKcs does not affect the CSR efficiency. Yet, the CSR junctions recovered from DNA-PKcs5A/5A B cells reveal increased chromosomal translocations, extensive use of distal switch regions (consistent with end resection), and preferential usage of microhomology—all signs of the alternative end-joining pathway. Thus, these results uncover a role of DNA-PKcs T2609 phosphorylation in promoting cNHEJ repair pathway choice during CSR.

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