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 Generation of a Mouse Model Lacking the Non-Homologous End-Joining Factor Mri/Cyren

2019 ◽  
Author(s):  
Sergio Castañeda-Zegarra ◽  
Camilla Huse ◽  
Øystein Røsand ◽  
Antonio Sarno ◽  
Mengtan Xing ◽  
...  
Keyword(s):  
Dna Repair ◽  
Class Switch Recombination ◽  
Physiological Role ◽  
Wild Type ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Mouse Development ◽  
Class Switch ◽  
Accessory Factors ◽  
Non Homologous End Joining

Classical non-homologous end joining (NHEJ) is a molecular pathway that detects, processes and ligates DNA double-strand breaks (DSBs) throughout the cell cycle. Mutations in several NHEJ genes result in neurological abnormalities and immunodeficiency both in humans and mice. The NHEJ pathway is required for the V(D)J recombination in developing B and T lymphocytes, and for class switch recombination in mature B cells. Ku heterodimer formed by Ku70 and Ku80 recognizes DSBs and facilitates the recruitment of accessory factors (e.g., DNA-PKcs, Artemis, Paxx and Mri/Cyren) and downstream core factors subunits XLF, XRCC4 and Lig4. Accessory factors might be dispensable for the process depending on the genetic background and DNA lesion type. To determine the physiological role of Mri in DNA repair and development, we introduced frame-shift mutation in the Mri gene in mice. We then analyzed the development of Mri-deficient mice as well as wild type and immunodeficient controls. Mice lacking Mri possessed reduced levels of class switch recombination in B lymphocytes and slow proliferation of neuronal progenitors when compared to wild type littermates. Human cell lines lacking Mri were as sensitive to DSBs as WT controls. Overall, we concluded that Mri/Cyren is largely dispensable for DNA repair and mouse development.

scholarly journals Generation of a Mouse Model Lacking the Non-Homologous End-Joining Factor Mri/Cyren

Biomolecules ◽  
2019 ◽  
Vol 9 (12) ◽  
pp. 798 ◽  
Author(s):  
Sergio Castañeda-Zegarra ◽  
Camilla Huse ◽  
Øystein Røsand ◽  
Antonio Sarno ◽  
Mengtan Xing ◽  
...  
Keyword(s):  
Dna Repair ◽  
Class Switch Recombination ◽  
Physiological Role ◽  
Wild Type ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Mouse Development ◽  
Class Switch ◽  
Accessory Factors ◽  
Non Homologous End Joining

Classical non-homologous end joining (NHEJ) is a molecular pathway that detects, processes, and ligates DNA double-strand breaks (DSBs) throughout the cell cycle. Mutations in several NHEJ genes result in neurological abnormalities and immunodeficiency both in humans and mice. The NHEJ pathway is required for V(D)J recombination in developing B and T lymphocytes, and for class switch recombination in mature B cells. The Ku heterodimer formed by Ku70 and Ku80 recognizes DSBs and facilitates the recruitment of accessory factors (e.g., DNA-PKcs, Artemis, Paxx and Mri/Cyren) and downstream core factor subunits X-ray repair cross-complementing group 4 (XRCC4), XRCC4-like factor (XLF), and DNA ligase 4 (Lig4). Accessory factors might be dispensable for the process, depending on the genetic background and DNA lesion type. To determine the physiological role of Mri in DNA repair and development, we introduced a frame-shift mutation in the Mri gene in mice. We then analyzed the development of Mri-deficient mice as well as wild type and immunodeficient controls. Mice lacking Mri possessed reduced levels of class switch recombination in B lymphocytes and slow proliferation of neuronal progenitors when compared to wild type littermates. Human cell lines lacking Mri were as sensitive to DSBs as the wild type controls. Overall, we concluded that Mri/Cyren is largely dispensable for DNA repair and mouse development.

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 Beyond DNA Repair: DNA-PKcs in Tumor Metastasis, Metabolism and Immunity

Cancers ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 3389
Author(s):  
Haitang Yang ◽  
Feng Yao ◽  
Thomas M. Marti ◽  
Ralph A. Schmid ◽  
Ren-Wang Peng
Keyword(s):  
Tumor Metastasis ◽  
Immune Escape ◽  
Critical Role ◽  
Large Body ◽  
Tumor Development ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Dependent Protein ◽  
Non Homologous End Joining

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a key component of the DNA-PK complex that has a well-characterized function in the non-homologous end-joining repair of DNA double-strand breaks. Since its identification, a large body of evidence has demonstrated that DNA-PKcs is frequently overexpressed in cancer, plays a critical role in tumor development and progression, and is associated with poor prognosis of cancer patients. Intriguingly, recent studies have suggested novel functions beyond the canonical role of DNA-PKcs, which has transformed the paradigm of DNA-PKcs in tumorigenesis and has reinvigorated the interest to target DNA-PKcs for cancer treatment. In this review, we update recent advances in DNA-PKcs, in particular the emerging roles in tumor metastasis, metabolic dysregulation, and immune escape. We further discuss the possible molecular basis that underpins the pleiotropism of DNA-PKcs in cancer. Finally, we outline the biomarkers that may predict the therapeutic response to DNA-PKcs inhibitor therapy. Understanding the functional repertoire of DNA-PKcs will provide mechanistic insights of DNA-PKcs in malignancy and, more importantly, may revolutionize the design and utility of DNA-PKcs-based precision cancer therapy.

scholarly journals Mapping of a Functional Recombination Motif that Defines Isotype Specificity for μ→γ3 Switch Recombination Implicates NF-κB p50 as the Isotype-specific Switching Factor

2004 ◽  
Vol 199 (5) ◽  
pp. 617-627 ◽  
Author(s):  
Amy L. Kenter ◽  
Robert Wuerffel ◽  
Carmen Dominguez ◽  
Ananth Shanmugam ◽  
Hongmei Zhang
Keyword(s):  
B Cells ◽  
Binding Site ◽  
Class Switch Recombination ◽  
Interleukin 4 ◽  
Specific Factor ◽  
Wild Type ◽  
Class Switch ◽  
Activation Induced Deaminase ◽  
Necessary And Sufficient

Ig class switch recombination (CSR) requires expression of activation-induced deaminase (AID) and production of germline transcripts to target S regions for recombination. However, the mechanism of CSR remains unclear. Here we show that an extrachromosomal S plasmid assay is AID dependent and that a single consensus repeat is both necessary and sufficient for isotype-specific CSR. Transfected switch substrates specific for μ→γ3 and μ→γ1 are stimulated to switch with lipopolysaccharide (LPS) alone or LPS and interleukin-4, respectively. An Sγ3/Sγ1 substrate containing only three Sγ3-associated nucleotides reconstituted LPS responsiveness and permitted mapping of a functional recombination motif specific for μ→γ3 CSR. This functional recombination motif colocalized with a binding site for NF-κB p50, and p50 binding to this site was previously established. We show a p50 requirement for plasmid-based μ→γ3 CSR using p50-deficient B cells. Switch junctions from p50-deficient B cells showed decreased lengths of microhomology between Sμ and Sγ3 relative to wild-type cells, indicating a function for p50 in the mechanics of CSR. We note a striking parallel between the affects of p50 and Msh2 deficiency on Sμ/Sγ3 junctions. The data suggest that p50 may be the isotype-specific factor in μ→γ3 CSR and epistatic with Msh2.

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 Ubiquitin-Specific-Processing Protease 47, A Novel 53BP1 regulator

2021 ◽  
Author(s):  
Doraid T. Sadideen ◽  
Baowei Chen ◽  
Manal Basili ◽  
Montaser Shaheen
Keyword(s):  
Strand Break ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Class Switch ◽  
Dna Double Strand Break ◽  
Radiation Induced ◽  
Non Homologous End Joining ◽  
Processing Protease

AbstractDNA double strand breaks (DSBs) are repair by homology-based repair or non-homologous end joining and multiple sub-pathways exist. 53BP1 is a key DNA double strand break repair protein that regulates repair pathway choice. It is key for joining DSBs during immunoglobulin heavy chain class switch recombination. Here we identify USP47 as a deubiquitylase that associates with and regulates 53BP1 function. USP47 loss results in 53BP1 instability in proteasome dependent manner, and defective 53BP1 ionizing radiation induced foci (IRIF). USP47 catalytic activity is required for maintaining 53BP1 protein level. Similar to 53BP1, USP47 depletion results in sensitivity to DNA DSB inducing agents and defective immunoglobulin CSR. Our findings establish a function for USP47 in DNA DSB repair at least partially through 53BP1.

scholarly journals An in vivo study of the impact of deficiency in the DNA repair proteins PAXX and XLF on development and maturation of the hemolymphoid system

2020 ◽  
Vol 295 (8) ◽  
pp. 2398-2406 ◽  
Author(s):  
Stefania Musilli ◽  
Vincent Abramowski ◽  
Benoit Roch ◽  
Jean-Pierre de Villartay
Keyword(s):  
Class Switch Recombination ◽  
Adaptive Immune System ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
Hematopoietic Progenitors ◽  
End Joining ◽  
Class Switch ◽  
A Genome ◽  
The Impact

Repair of DNA double-strand breaks by the nonhomologous end joining pathway is central for proper development of the adaptive immune system. This repair pathway involves eight factors, including XRCC4-like factor (XLF)/Cernunnos and the paralog of XRCC4 and XLF, PAXX nonhomologous end joining factor (PAXX). Xlf−/− and Paxx−/− mice are viable and exhibit only a mild immunophenotype. However, mice lacking both PAXX and XLF are embryonic lethal because postmitotic neurons undergo massive apoptosis in embryos. To decipher the roles of PAXX and XLF in both variable, diversity, and joining recombination and immunoglobulin class switch recombination, here, using Cre/lox-specific deletion to prevent double-KO embryonic lethality, we developed two mouse models of a conditional Xlf KO in a Paxx−/− background. Cre expressed under control of the iVav or CD21 promoter enabled Xlf deletion in early hematopoietic progenitors and splenic mature B cells, respectively. We demonstrate the XLF and PAXX interplay during variable, diversity, and joining recombination in vivo but not during class switch recombination, for which PAXX appeared to be fully dispensable. Xlf/Paxx double KO in hematopoietic progenitors resulted in a shorter lifespan associated with onset of thymic lymphomas, revealing a genome caretaking function of XLF/PAXX.

scholarly journals AZD7648 is a potent and selective DNA-PK inhibitor that enhances radiation, chemotherapy and olaparib activity

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Jacqueline H. L. Fok ◽  
Antonio Ramos-Montoya ◽  
Mercedes Vazquez-Chantada ◽  
Paul W. G. Wijnhoven ◽  
Valeria Follia ◽  
...  
Keyword(s):  
Dna Damage ◽  
Clinical Investigation ◽  
Parp Inhibitor ◽  
Tumour Regression ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Current Therapies ◽  
Dependent Protein ◽  
Non Homologous End Joining ◽  
Pdx Models

Abstract DNA-dependent protein kinase (DNA-PK) is a critical player in the DNA damage response (DDR) and instrumental in the non-homologous end-joining pathway (NHEJ) used to detect and repair DNA double-strand breaks (DSBs). We demonstrate that the potent and highly selective DNA-PK inhibitor, AZD7648, is an efficient sensitizer of radiation- and doxorubicin-induced DNA damage, with combinations in xenograft and patient-derived xenograft (PDX) models inducing sustained regressions. Using ATM-deficient cells, we demonstrate that AZD7648, in combination with the PARP inhibitor olaparib, increases genomic instability, resulting in cell growth inhibition and apoptosis. AZD7648 enhanced olaparib efficacy across a range of doses and schedules in xenograft and PDX models, enabling sustained tumour regression and providing a clear rationale for its clinical investigation. Through its differentiated mechanism of action as an NHEJ inhibitor, AZD7648 complements the current armamentarium of DDR-targeted agents and has potential in combination with these agents to achieve deeper responses to current therapies.

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.

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