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 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 53BP1 is required for class switch recombination

2004 ◽  
Vol 165 (4) ◽  
pp. 459-464 ◽  
Author(s):  
Irene M. Ward ◽  
Bernardo Reina-San-Martin ◽  
Alexandru Olaru ◽  
Kay Minn ◽  
Koji Tamada ◽  
...  
Keyword(s):  
Class Switch Recombination ◽  
Cell Cycle Checkpoint ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Checkpoint Control ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Class Switch ◽  
Cycle Checkpoint

53BP1 participates early in the DNA damage response and is involved in cell cycle checkpoint control. Moreover, the phenotype of mice and cells deficient in 53BP1 suggests a defect in DNA repair (Ward et al., 2003b). Therefore, we asked whether or not 53BP1 would be required for the efficient repair of DNA double strand breaks. Our data indicate that homologous recombination by gene conversion does not depend on 53BP1. Moreover, 53BP1-deficient mice support normal V(D)J recombination, indicating that 53BP1 is not required for “classic” nonhomologous end joining. However, class switch recombination is severely impaired in the absence of 53BP1, suggesting that 53BP1 facilitates DNA end joining in a way that is not required or redundant for the efficient closing of RAG-induced strand breaks. These findings are similar to those observed in mice or cells deficient in the tumor suppressors ATM and H2AX, further suggesting that the functions of ATM, H2AX, and 53BP1 are closely linked.

scholarly journals CoincidentIn VitroAnalysis of DNA-PK-Dependent and -Independent Nonhomologous End Joining

2010 ◽  
Vol 2010 ◽  
pp. 1-11 ◽  
Author(s):  
Cynthia L. Hendrickson ◽  
Shubhadeep Purkayastha ◽  
Elzbieta Pastwa ◽  
Ronald D. Neumann ◽  
Thomas A. Winters
Keyword(s):  
Mammalian Cells ◽  
Current Model ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Ligase Iv ◽  
Dependent Protein ◽  
Regulatory Functions

In mammalian cells, DNA double-strand breaks (DSBs) are primarily repaired by nonhomologous end joining (NHEJ). The current model suggests that the Ku 70/80 heterodimer binds to DSB ends and recruits DNA-PKcsto form the active DNA-dependent protein kinase, DNA-PK. Subsequently, XRCC4, DNA ligase IV, XLF and most likely, other unidentified components participate in the final DSB ligation step. Therefore, DNA-PK plays a key role in NHEJ due to its structural and regulatory functions that mediate DSB end joining. However, recent studies show that additional DNA-PK-independent NHEJ pathways also exist. Unfortunately, the presence of DNA-PKcsappears to inhibit DNA-PK-independent NHEJ, andin vitroanalysis of DNA-PK-independent NHEJ in the presence of the DNA-PKcsprotein remains problematic. We have developed anin vitroassay that is preferentially active for DNA-PK-independent DSB repair based solely on its reaction conditions, facilitating coincident differential biochemical analysis of the two pathways. The results indicate the biochemically distinct nature of the end-joining mechanisms represented by the DNA-PK-dependent and -independent NHEJ assays as well as functional differences between the two pathways.

scholarly journals Ku Heterodimer-Independent End Joining in Trypanosoma brucei Cell Extracts Relies upon Sequence Microhomology

2007 ◽  
Vol 6 (10) ◽  
pp. 1773-1781 ◽  
Author(s):  
Peter Burton ◽  
David J. McBride ◽  
Jonathan M. Wilkes ◽  
J. David Barry ◽  
Richard McCulloch
Keyword(s):  
Homologous Recombination ◽  
Trypanosoma Brucei ◽  
Antigenic Variation ◽  
Bacterial Species ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Cell Extracts ◽  
Ligase Iv

ABSTRACT DNA double-strand breaks (DSBs) are repaired primarily by two distinct pathways: homologous recombination and nonhomologous end joining (NHEJ). NHEJ has been found in all eukaryotes examined to date and has been described recently for some bacterial species, illustrating its ancestry. Trypanosoma brucei is a divergent eukaryotic protist that evades host immunity by antigenic variation, a process in which homologous recombination plays a crucial function. While homologous recombination has been examined in some detail in T. brucei, little work has been done to examine what other DSB repair pathways the parasite utilizes. Here we show that T. brucei cell extracts support the end joining of linear DNA molecules. These reactions are independent of the Ku heterodimer, indicating that they are distinct from NHEJ, and are guided by sequence microhomology. We also demonstrate bioinformatically that T. brucei, in common with other kinetoplastids, does not encode recognizable homologues of DNA ligase IV or XRCC4, suggesting that NHEJ is either absent or mechanistically diverged in these pathogens.

scholarly journals Parp3 promotes long-range end joining in murine cells

2018 ◽  
Vol 115 (40) ◽  
pp. 10076-10081 ◽  
Author(s):  
Jacob V. Layer ◽  
J. Patrick Cleary ◽  
Alexander J. Brown ◽  
Kristen E. Stevenson ◽  
Sara N. Morrow ◽  
...  
Keyword(s):  
Chromosomal Rearrangements ◽  
Embryonic Stem ◽  
Cell Types ◽  
Strand Break ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Class Switch

Chromosomal rearrangements, including translocations, are early and essential events in the formation of many tumors. Previous studies that defined the genetic requirements for rearrangement formation have identified differences between murine and human cells, most notably in the role of classic and alternative nonhomologous end-joining (NHEJ) factors. We reported that poly(ADP)ribose polymerase 3 (PARP3) promotes chromosomal rearrangements induced by endonucleases in multiple human cell types. We show here that in contrast to classic (c-NHEJ) factors, Parp3 also promotes rearrangements in murine cells, including translocations in murine embryonic stem cells (mESCs), class–switch recombination in primary B cells, and inversions in tail fibroblasts that generateEml4–Alkfusions. In mESCs, Parp3-deficient cells had shorter deletion lengths at translocation junctions. This was corroborated using next-generation sequencing ofEml4–Alkjunctions in tail fibroblasts and is consistent with a role for Parp3 in promoting the processing of DNA double-strand breaks. We confirmed a previous report that Parp1 also promotes rearrangement formation. In contrast with Parp3, rearrangement junctions in the absence of Parp1 had longer deletion lengths, suggesting that Parp1 may suppress double-strand break processing. Together, these data indicate that Parp3 and Parp1 promote rearrangements with distinct phenotypes.

scholarly journals Unexpected behavior of DNA polymerase Mu opposite template 8-oxo-7,8-dihydro-2′-guanosine

2019 ◽  
Vol 47 (17) ◽  
pp. 9410-9422 ◽  
Author(s):  
Andrea M Kaminski ◽  
Kishore K Chiruvella ◽  
Dale A Ramsden ◽  
Thomas A Kunkel ◽  
Katarzyna Bebenek ◽  
...  
Keyword(s):  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Steady State Kinetics ◽  
Ligase Iv ◽  
Template Dna ◽  
Dna Substrate

Abstract DNA double-strand breaks (DSBs) resulting from reactive oxygen species generated by exposure to UV and ionizing radiation are characterized by clusters of lesions near break sites. Such complex DSBs are repaired slowly, and their persistence can have severe consequences for human health. We have therefore probed DNA break repair containing a template 8-oxo-7,8-dihydro-2′-guanosine (8OG) by Family X Polymerase μ (Pol μ) in steady-state kinetics and cell-based assays. Pol μ tolerates 8OG-containing template DNA substrates, and the filled products can be subsequently ligated by DNA Ligase IV during Nonhomologous end-joining. Furthermore, Pol μ exhibits a strong preference for mutagenic bypass of 8OG by insertion of adenine. Crystal structures reveal that the template 8OG is accommodated in the Pol μ active site with none of the DNA substrate distortions observed for Family X siblings Pols β or λ. Kinetic characterization of template 8OG bypass indicates that Pol μ inserts adenosine nucleotides with weak sugar selectivity and, given the high cellular concentration of ATP, likely performs its role in repair of complex 8OG-containing DSBs using ribonucleotides.

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.

Pluronic copolymer encapsulated SCR7 as a potential anticancer agent

2015 ◽  
Vol 177 ◽  
pp. 155-161 ◽  
Author(s):  
Franklin John ◽  
Jinu George ◽  
Mrinal Srivastava ◽  
P. A. Hassan ◽  
V. K. Aswal ◽  
...  
Keyword(s):  
Therapeutic Agent ◽  
Analytical Techniques ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Physical Interaction ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Anticancer Properties

Nonhomologous end joining (NHEJ) of DNA double strand breaks (DSBs) inside cells can be selectively inhibited by 5,6-bis-(benzylideneamino)-2-mercaptopyrimidin-4-ol (SCR7) which possesses anticancer properties. The hydrophobicity of SCR7 decreases its bioavailability which is a major setback in the utilization of this compound as a therapeutic agent. In order to circumvent the drawback of SCR7, we prepared a polymer encapsulated form of SCR7. The physical interaction of SCR7 and Pluronic® copolymer is evident from different analytical techniques. The in vitro cytotoxicity of the drug formulations is established using the MTT assay.

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 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.

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