Analyses of TCRB rearrangements substantiate a profound deficit in recombination signal sequence joining in SCID foals: implications for the role of DNA-dependent protein kinase in V(D)J recombination.
Abstract: We reported previously that the genetic SCID disease observed in Arabian foals is explained by a defect in V(D)J recombination that profoundly affects both coding and signal end joining. As in C.B-17 SCID mice, the molecular defect in SCID foals is in the catalytic subunit of the DNA-dependent protein kinase (DNA-PKCS); however, in SCID mice, signal end resolution remains relatively intact. Moreover, recent reports indicate that mice that completely lack DNA-PKCS also generate signal joints at levels that are indistinguishable from those observed in C.B-17 SCID mice, eliminating the possibility that a partially active version of DNA-PKCS facilitates signal end resolution in SCID mice. We have analyzed TCRB rearrangements and find that signal joints are reduced by approximately 4 logs in equine SCID thymocytes as compared with normal horse thymocytes. A potential explanation for the differences between SCID mice and foals is that the mutant DNA-PKCS allele in SCID foals inhibits signal end resolution. We tested this hypothesis using DNA-PKCS expression vectors; in sum, we find no evidence of a dominant-negative effect by the mutant protein. These and other recent data are consistent with an emerging consensus: that in normal cells, DNA-PKCS participates in both coding and signal end resolution, but in the absence of DNA-PKCS an undefined end joining pathway (which is variably expressed in different species and cell types) can facilitate imperfect signal and coding end joining.
Publication Date: 2000-01-21 PubMed ID: 10640757DOI: 10.4049/jimmunol.164.3.1416Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
- Research Support
- U.S. Gov't
- P.H.S.
Summary
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This research article investigates the role of a certain protein in a genetic disease seen in Arabian foals, known as Severe Combined Immunodeficiency (SCID). The researchers analyze specific component rearrangements in relation to the disease, providing evidence that suggest differences in how the disease presents in different species.
Introduction and Background
- The study is based on previous research which established the genetic disease SCID in Arabian foals is due to a defect in a particular genetic recombination (V(D)J), significantly affecting both coding and signal end joining, essential processes for immune development.
- In both SCID foals and a strain of SCID mice, the molecular defect lies in the catalytic subunit of the DNA-dependent protein kinase (DNA-PKCS) – an enzyme vital for DNA repair and V(D)J recombination. The difference is that in SCID mice, the resolution of the signal end remains relatively more intact.
Research Aims and Methodology
- The researchers examined T-cell receptor beta (TCRB) rearrangements and found that the level of signal joint formation in SCID foal thymocytes were drastically reduced compared to normal foal thymocytes (about a 10,000-fold reduction).
- To explain the difference between SCID foals and mice, the authors suggested that the mutant DNA-PKCS allele in SCID foals might inhibit signal end resolution. They investigated this hypothesis by using DNA-PKCS expression vectors.
Findings and Conclusions
- The results did not support the hypothesis. There was no evidence found that the mutant protein has a dominant-negative effect.
- The authors concluded that the DNA-PKCS is involved in signal and coding end resolution in normal cells, and without it, another undefined end joining route—varying in different species and cell types—might facilitate imperfect signal and coding end joining.
Cite This Article
APA
Shin EK, Rijkers T, Pastink A, Meek K.
(2000).
Analyses of TCRB rearrangements substantiate a profound deficit in recombination signal sequence joining in SCID foals: implications for the role of DNA-dependent protein kinase in V(D)J recombination.
J Immunol, 164(3), 1416-1424.
https://doi.org/10.4049/jimmunol.164.3.1416 Publication
Researcher Affiliations
- Harold C. Simmons Arthritis Research Center, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75235, USA.
MeSH Terms
- Alleles
- Animals
- Catalytic Domain / genetics
- Cell Line
- Codon / analysis
- Codon / genetics
- Codon / immunology
- DNA Nucleotidyltransferases / antagonists & inhibitors
- DNA Nucleotidyltransferases / genetics
- DNA-Activated Protein Kinase
- DNA-Binding Proteins
- Gene Rearrangement, beta-Chain T-Cell Antigen Receptor
- Horses
- Integrases
- Introns / genetics
- Introns / immunology
- Mutation
- Protein Serine-Threonine Kinases / deficiency
- Protein Serine-Threonine Kinases / genetics
- Protein Serine-Threonine Kinases / physiology
- Receptors, Antigen, T-Cell, alpha-beta / genetics
- Recombinases
- Recombination, Genetic / immunology
- Severe Combined Immunodeficiency / enzymology
- Severe Combined Immunodeficiency / genetics
- Severe Combined Immunodeficiency / immunology
- Signal Transduction / genetics
- Signal Transduction / immunology
- T-Lymphocytes / enzymology
- T-Lymphocytes / immunology
- T-Lymphocytes / pathology
Grant Funding
- R01 AI048758 / NIAID NIH HHS
- AI32600 / NIAID NIH HHS
Citations
This article has been cited 18 times.- Britton S, Chanut P, Delteil C, Barboule N, Frit P, Calsou P. ATM antagonizes NHEJ proteins assembly and DNA-ends synapsis at single-ended DNA double strand breaks. Nucleic Acids Res 2020 Sep 25;48(17):9710-9723.
- Neal JA, Meek K. Deciphering phenotypic variance in different models of DNA-PKcs deficiency. DNA Repair (Amst) 2019 Jan;73:7-16.
- Meek K, Xu Y, Bailie C, Yu K, Neal JA. The ATM Kinase Restrains Joining of Both VDJ Signal and Coding Ends. J Immunol 2016 Oct 15;197(8):3165-3174.
- Neal JA, Xu Y, Abe M, Hendrickson E, Meek K. Restoration of ATM Expression in DNA-PKcs-Deficient Cells Inhibits Signal End Joining. J Immunol 2016 Apr 1;196(7):3032-42.
- Neal JA, Sugiman-Marangos S, VanderVere-Carozza P, Wagner M, Turchi J, Lees-Miller SP, Junop MS, Meek K. Unraveling the complexities of DNA-dependent protein kinase autophosphorylation. Mol Cell Biol 2014 Jun;34(12):2162-75.
- Woodbine L, Neal JA, Sasi NK, Shimada M, Deem K, Coleman H, Dobyns WB, Ogi T, Meek K, Davies EG, Jeggo PA. PRKDC mutations in a SCID patient with profound neurological abnormalities. J Clin Invest 2013 Jul;123(7):2969-80.
- Gapud EJ, Sleckman BP. Unique and redundant functions of ATM and DNA-PKcs during V(D)J recombination. Cell Cycle 2011 Jun 15;10(12):1928-35.
- Neal JA, Dang V, Douglas P, Wold MS, Lees-Miller SP, Meek K. Inhibition of homologous recombination by DNA-dependent protein kinase requires kinase activity, is titratable, and is modulated by autophosphorylation. Mol Cell Biol 2011 Apr;31(8):1719-33.
- Gapud EJ, Dorsett Y, Yin B, Callen E, Bredemeyer A, Mahowald GK, Omi KQ, Walker LM, Bednarski JJ, McKinnon PJ, Bassing CH, Nussenzweig A, Sleckman BP. Ataxia telangiectasia mutated (Atm) and DNA-PKcs kinases have overlapping activities during chromosomal signal joint formation. Proc Natl Acad Sci U S A 2011 Feb 1;108(5):2022-7.
- Liu J, Naegele JR, Lin SL. The DNA-PK catalytic subunit regulates Bax-mediated excitotoxic cell death by Ku70 phosphorylation. Brain Res 2009 Nov 3;1296:164-75.
- Douglas P, Cui X, Block WD, Yu Y, Gupta S, Ding Q, Ye R, Morrice N, Lees-Miller SP, Meek K. The DNA-dependent protein kinase catalytic subunit is phosphorylated in vivo on threonine 3950, a highly conserved amino acid in the protein kinase domain. Mol Cell Biol 2007 Mar;27(5):1581-91.
- Gupta S, Meek K. The leucine rich region of DNA-PKcs contributes to its innate DNA affinity. Nucleic Acids Res 2005;33(22):6972-81.
- Cui X, Yu Y, Gupta S, Cho YM, Lees-Miller SP, Meek K. Autophosphorylation of DNA-dependent protein kinase regulates DNA end processing and may also alter double-strand break repair pathway choice. Mol Cell Biol 2005 Dec;25(24):10842-52.
- Convery E, Shin EK, Ding Q, Wang W, Douglas P, Davis LS, Nickoloff JA, Lees-Miller SP, Meek K. Inhibition of homologous recombination by variants of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs). Proc Natl Acad Sci U S A 2005 Feb 1;102(5):1345-50.
- Ding Q, Reddy YV, Wang W, Woods T, Douglas P, Ramsden DA, Lees-Miller SP, Meek K. Autophosphorylation of the catalytic subunit of the DNA-dependent protein kinase is required for efficient end processing during DNA double-strand break repair. Mol Cell Biol 2003 Aug;23(16):5836-48.
- Woods T, Wang W, Convery E, Errami A, Zdzienicka MZ, Meek K. A single amino acid substitution in DNA-PKcs explains the novel phenotype of the CHO mutant, XR-C2. Nucleic Acids Res 2002 Dec 1;30(23):5120-8.
- Kienker LJ, Shin EK, Meek K. Both V(D)J recombination and radioresistance require DNA-PK kinase activity, though minimal levels suffice for V(D)J recombination. Nucleic Acids Res 2000 Jul 15;28(14):2752-61.
- Pascarella G, Conner KN, Goff NJ, Carninci P, Olive AJ, Meek K. Compared to other NHEJ factors, DNA-PK protein and RNA levels are markedly increased in all higher primates, but not in prosimians or other mammals. DNA Repair (Amst) 2024 Oct;142:103737.
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