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PMID: 19450527 Published · ppublish English Journal Article Research Support, N.I.H., Intramural Research Support, Non-U.S. Gov't

Essential role for DNA-PKcs in DNA double-strand break repair and apoptosis in ATM-deficient lymphocytes.

Molecular cell ·Vol. 34 ·No. 3 ·2009-05-15 ·Pages 285-97

Callén E, Jankovic M, Wong N, Zha S, Chen HT, Difilippantonio S, Di Virgilio M, Heidkamp G, Alt FW, Nussenzweig A, Nussenzweig M

Abstract

The DNA double-strand break (DSB) repair protein DNA-PKcs and the signal transducer ATM are both activated by DNA breaks and phosphorylate similar substrates in vitro, yet appear to have distinct functions in vivo. Here, we show that ATM and DNA-PKcs have overlapping functions in lymphocytes. Ablation of both kinase activities in cells undergoing immunoglobulin class switch recombination leads to a compound defect in switching and a synergistic increase in chromosomal fragmentation, DNA insertions, and translocations due to aberrant processing of DSBs. These abnormalities are attributed to a compound deficiency in phosphorylation of key proteins required for DNA repair, class switching, and cell death. Notably, both kinases are required for normal levels of p53 phosphorylation in B and T cells and p53-dependent apoptosis. Our experiments reveal a DNA-PKcs-dependent pathway that regulates DNA repair and activation of p53 in the absence of ATM.

MeSH Terms
Animals Apoptosis/physiology Ataxia Telangiectasia Mutated Proteins Base Sequence Cell Cycle Proteins/genetics,metabolism Cells, Cultured DNA Breaks, Double-Stranded DNA Repair DNA-Activated Protein Kinase/genetics,metabolism DNA-Binding Proteins/genetics,metabolism Fibroblasts/cytology,physiology Genomic Instability Immunoglobulin Class Switching Lymphocytes/cytology,physiology Mice Mice, Knockout Molecular Sequence Data Nuclear Proteins/genetics,metabolism Protein Serine-Threonine Kinases/genetics,metabolism Repressor Proteins/genetics,metabolism Thymus Gland/cytology Tripartite Motif-Containing Protein 28 Tumor Suppressor Protein p53/genetics,metabolism Tumor Suppressor Proteins/genetics,metabolism
Chemicals
Cell Cycle Proteins DNA-Binding Proteins Nuclear Proteins Repressor Proteins Tumor Suppressor Protein p53 Tumor Suppressor Proteins Trim28 protein, mouse Tripartite Motif-Containing Protein 28 Ataxia Telangiectasia Mutated Proteins Atm protein, mouse DNA-Activated Protein Kinase Prkdc protein, mouse Protein Serine-Threonine Kinases
Authors & Affiliations
11 authors, click to expand affiliations / ORCID
Callén Elsa
Experimental Immunology Branch, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
Jankovic Mila
Wong Nancy
Zha Shan
Chen Hua-Tang
Difilippantonio Simone
Di Virgilio Michela
Heidkamp Gordon
Alt Frederick W
Nussenzweig André
Nussenzweig Michel
References (87)
87 references, click to expand
  1. Immunoglobulin class switch recombination is impaired in Atm-deficient mice.
    J Exp Med. 2004 Nov 1;200(9):1111-21 PMID: 15504820
  2. 53BP1 links DNA damage-response pathways to immunoglobulin heavy chain class-switch recombination.
    Nat Immunol. 2004 May;5(5):481-7 PMID: 15077110
  3. ATM- and cell cycle-dependent regulation of ATR in response to DNA double-strand breaks.
    Nat Cell Biol. 2006 Jan;8(1):37-45 PMID: 16327781
  4. Identification of a highly potent and selective DNA-dependent protein kinase (DNA-PK) inhibitor (NU7441) by screening of chromenone libraries.
    Bioorg Med Chem Lett. 2004 Dec 20;14(24):6083-7 PMID: 15546735
  5. Complementary functions of ATM and H2AX in development and suppression of genomic instability.
    Proc Natl Acad Sci U S A. 2008 Jul 8;105(27):9302-6 PMID: 18599436
  6. The DNA-dependent protein kinase: requirement for DNA ends and association with Ku antigen.
    Cell. 1993 Jan 15;72(1):131-42 PMID: 8422676
  7. The DNA-dependent protein kinase.
    Genes Dev. 1999 Apr 15;13(8):916-34 PMID: 10215620
  8. AID is required to initiate Nbs1/gamma-H2AX focus formation and mutations at sites of class switching.
    Nature. 2001 Dec 6;414(6864):660-665 PMID: 11740565
  9. AID to overcome the limitations of genomic information.
    Nat Immunol. 2005 Jul;6(7):655-61 PMID: 15970938
  10. Nibrin functions in Ig class-switch recombination.
    Proc Natl Acad Sci U S A. 2005 Feb 1;102(5):1584-9 PMID: 15668383
  11. AID is required for c-myc/IgH chromosome translocations in vivo.
    Cell. 2004 Aug 20;118(4):431-8 PMID: 15315756
  12. ATR: an essential regulator of genome integrity.
    Nat Rev Mol Cell Biol. 2008 Aug;9(8):616-27 PMID: 18594563
  13. AID mediates hypermutation by deaminating single stranded DNA.
    J Exp Med. 2003 May 19;197(10):1291-6 PMID: 12756266
  14. Autophosphorylation of DNA-PKCS regulates its dynamics at DNA double-strand breaks.
    J Cell Biol. 2007 Apr 23;177(2):219-29 PMID: 17438073
  15. Genomic instability, endoreduplication, and diminished Ig class-switch recombination in B cells lacking Nbs1.
    Proc Natl Acad Sci U S A. 2005 Feb 1;102(5):1590-5 PMID: 15668392
  16. H2AX is required for recombination between immunoglobulin switch regions but not for intra-switch region recombination or somatic hypermutation.
    J Exp Med. 2003 Jun 16;197(12):1767-78 PMID: 12810694
  17. ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage.
    Science. 2007 May 25;316(5828):1160-6 PMID: 17525332
  18. p53 heterozygosity alters the mRNA expression of p53 target genes in the bone marrow in response to inhaled benzene.
    Toxicol Sci. 2002 Apr;66(2):209-15 PMID: 11896287
  19. ATM prevents the persistence and propagation of chromosome breaks in lymphocytes.
    Cell. 2007 Jul 13;130(1):63-75 PMID: 17599403
  20. Transcription-targeted DNA deamination by the AID antibody diversification enzyme.
    Nature. 2003 Apr 17;422(6933):726-30 PMID: 12692563
  21. A comprehensive study of p53 transcriptional activity in thymus and spleen of gamma irradiated mouse: high sensitivity of genes involved in the two main apoptotic pathways.
    Int J Radiat Biol. 2006 Nov;82(11):761-70 PMID: 17148260
  22. Conserved modes of recruitment of ATM, ATR and DNA-PKcs to sites of DNA damage.
    Nature. 2005 Mar 31;434(7033):605-11 PMID: 15758953
  23. Radiosensitization and DNA repair inhibition by the combined use of novel inhibitors of DNA-dependent protein kinase and poly(ADP-ribose) polymerase-1.
    Cancer Res. 2003 Sep 15;63(18):6008-15 PMID: 14522929
  24. Specific expression of activation-induced cytidine deaminase (AID), a novel member of the RNA-editing deaminase family in germinal center B cells.
    J Biol Chem. 1999 Jun 25;274(26):18470-6 PMID: 10373455
  25. AID mutates E. coli suggesting a DNA deamination mechanism for antibody diversification.
    Nature. 2002 Jul 4;418(6893):99-103 PMID: 12097915
  26. Ku autoantigen is the regulatory component of a template-associated protein kinase that phosphorylates RNA polymerase II.
    Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):11920-4 PMID: 1465419
  27. Essential and dispensable roles of ATR in cell cycle arrest and genome maintenance.
    Genes Dev. 2003 Mar 1;17(5):615-28 PMID: 12629044
  28. Targeting AID to the Ig genes.
    Adv Exp Med Biol. 2007;596:93-109 PMID: 17338179
  29. Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes.
    Science. 2003 Jun 6;300(5625):1542-8 PMID: 12791985
  30. Rapid activation of ATM on DNA flanking double-strand breaks.
    Nat Cell Biol. 2007 Nov;9(11):1311-8 PMID: 17952060
  31. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation.
    Nature. 2003 Jan 30;421(6922):499-506 PMID: 12556884
  32. Phosphorylation of SMC1 is a critical downstream event in the ATM-NBS1-BRCA1 pathway.
    Genes Dev. 2004 Jun 15;18(12):1423-38 PMID: 15175241
  33. Evolution of the immunoglobulin heavy chain class switch recombination mechanism.
    Adv Immunol. 2007;94:157-214 PMID: 17560275
  34. IgH class switch recombination to IgG1 in DNA-PKcs-deficient B cells.
    Immunity. 2002 Apr;16(4):607-17 PMID: 11970883
  35. 53BP1 is required for class switch recombination.
    J Cell Biol. 2004 May 24;165(4):459-64 PMID: 15159415
  36. Genetic interactions between ATM and the nonhomologous end-joining factors in genomic stability and development.
    Proc Natl Acad Sci U S A. 2001 Mar 13;98(6):3243-8 PMID: 11248063
  37. Synthetic lethality between mutation in Atm and DNA-PK(cs) during murine embryogenesis.
    Curr Biol. 2001 Feb 6;11(3):191-4 PMID: 11231155
  38. Rapid activation of ATR by ionizing radiation requires ATM and Mre11.
    J Biol Chem. 2006 Apr 7;281(14):9346-50 PMID: 16431910
  39. Interfaces between the detection, signaling, and repair of DNA damage.
    Science. 2002 Jul 26;297(5581):547-51 PMID: 12142523
  40. Transcription enhances AID-mediated cytidine deamination by exposing single-stranded DNA on the nontemplate strand.
    Nat Immunol. 2003 May;4(5):452-6 PMID: 12692548
  41. AID is required for the chromosomal breaks in c-myc that lead to c-myc/IgH translocations.
    Cell. 2008 Dec 12;135(6):1028-38 PMID: 19070574
  42. Genomic instability in mice lacking histone H2AX.
    Science. 2002 May 3;296(5569):922-7 PMID: 11934988
  43. Activation-induced cytidine deaminase-dependent DNA breaks in class switch recombination occur during G1 phase of the cell cycle and depend upon mismatch repair.
    J Immunol. 2007 Nov 1;179(9):6064-71 PMID: 17947680
  44. Sensitization of breast carcinoma cells to ionizing radiation by small molecule inhibitors of DNA-dependent protein kinase and ataxia telangiectsia mutated.
    Biochem Pharmacol. 2005 Dec 19;71(1-2):13-20 PMID: 16293233
  45. Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme.
    Cell. 2000 Sep 1;102(5):553-63 PMID: 11007474
  46. Requirement for the kinase activity of human DNA-dependent protein kinase catalytic subunit in DNA strand break rejoining.
    Mol Cell Biol. 1999 May;19(5):3877-84 PMID: 10207111
  47. Chromatin relaxation in response to DNA double-strand breaks is modulated by a novel ATM- and KAP-1 dependent pathway.
    Nat Cell Biol. 2006 Aug;8(8):870-6 PMID: 16862143
  48. Distinct domains in Nbs1 regulate irradiation-induced checkpoints and apoptosis.
    J Exp Med. 2007 May 14;204(5):1003-11 PMID: 17485521
  49. DNA-dependent protein kinase activity is not required for immunoglobulin class switching.
    J Exp Med. 2002 Dec 2;196(11):1483-95 PMID: 12461083
  50. H2AX prevents DNA breaks from progressing to chromosome breaks and translocations.
    Mol Cell. 2006 Jan 20;21(2):201-14 PMID: 16427010
  51. ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex.
    Science. 2005 Apr 22;308(5721):551-4 PMID: 15790808
  52. Antigen receptor diversification and chromosome translocations.
    Nat Immunol. 2007 Aug;8(8):801-8 PMID: 17641661
  53. Molecular mechanisms of antibody somatic hypermutation.
    Annu Rev Biochem. 2007;76:1-22 PMID: 17328676
  54. Replication protein A2 phosphorylation after DNA damage by the coordinated action of ataxia telangiectasia-mutated and DNA-dependent protein kinase.
    Cancer Res. 2001 Dec 1;61(23):8554-63 PMID: 11731442
  55. ATM regulates ATR chromatin loading in response to DNA double-strand breaks.
    J Exp Med. 2006 Feb 20;203(2):297-303 PMID: 16461339
  56. ATM and DNA-PK function redundantly to phosphorylate H2AX after exposure to ionizing radiation.
    Cancer Res. 2004 Apr 1;64(7):2390-6 PMID: 15059890
  57. DNA-dependent protein kinase is not required for accumulation of p53 or cell cycle arrest after DNA damage.
    Cancer Res. 1997 Jan 1;57(1):68-74 PMID: 8988043
  58. Ataxia telangiectasia mutated (ATM) is essential for DNA-PKcs phosphorylations at the Thr-2609 cluster upon DNA double strand break.
    J Biol Chem. 2007 Mar 2;282(9):6582-7 PMID: 17189255
  59. ATM and related protein kinases: safeguarding genome integrity.
    Nat Rev Cancer. 2003 Mar;3(3):155-68 PMID: 12612651
  60. Chk2 is a tumor suppressor that regulates apoptosis in both an ataxia telangiectasia mutated (ATM)-dependent and an ATM-independent manner.
    Mol Cell Biol. 2002 Sep;22(18):6521-32 PMID: 12192050
  61. The catalytic subunit of DNA-protein kinase (DNA-PKcs) is not required for Ig class-switch recombination.
    Proc Natl Acad Sci U S A. 2007 Feb 20;104(8):2843-8 PMID: 17296939
  62. 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 PMID: 17158925
  63. Processive AID-catalysed cytosine deamination on single-stranded DNA simulates somatic hypermutation.
    Nature. 2003 Jul 3;424(6944):103-7 PMID: 12819663
  64. Reduced switching in SCID B cells is associated with altered somatic mutation of recombined S regions.
    J Immunol. 2003 Dec 15;171(12):6556-64 PMID: 14662857
  65. Requirement for Atm in ionizing radiation-induced cell death in the developing central nervous system.
    Science. 1998 May 15;280(5366):1089-91 PMID: 9582124
  66. ATM is required for efficient recombination between immunoglobulin switch regions.
    J Exp Med. 2004 Nov 1;200(9):1103-10 PMID: 15520243
  67. DNA-dependent protein kinase is not required for the p53-dependent response to DNA damage.
    Nature. 1999 Jul 1;400(6739):81-3 PMID: 10403253
  68. Activation-induced deaminase: controversies and open questions.
    Trends Immunol. 2005 Feb;26(2):90-6 PMID: 15668124
  69. 53BP1 facilitates long-range DNA end-joining during V(D)J recombination.
    Nature. 2008 Nov 27;456(7221):529-33 PMID: 18931658
  70. Synapsis of DNA ends by DNA-dependent protein kinase.
    EMBO J. 2002 Jun 17;21(12):3192-200 PMID: 12065431
  71. Autophosphorylation at serine 1987 is dispensable for murine Atm activation in vivo.
    Nature. 2006 Sep 14;443(7108):222-5 PMID: 16906133
  72. Role of genomic instability and p53 in AID-induced c-myc-Igh translocations.
    Nature. 2006 Mar 2;440(7080):105-9 PMID: 16400328
  73. Ataxia-telangiectasia and Rad3-related and DNA-dependent protein kinase cooperate in G2 checkpoint activation by the DNA strand-breaking nucleoside analogue 2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine.
    Mol Cancer Ther. 2008 Jan;7(1):133-42 PMID: 18202016
  74. The p53 response to DNA damage in vivo is independent of DNA-dependent protein kinase.
    Mol Cell Biol. 2000 Jun;20(11):4075-83 PMID: 10805749
  75. DNA-dependent protein kinase-independent activation of p53 in response to DNA damage.
    J Biol Chem. 1999 Jun 11;274(24):17139-43 PMID: 10358069
  76. Regulation of hypermutation by activation-induced cytidine deaminase phosphorylation.
    Proc Natl Acad Sci U S A. 2006 Jun 6;103(23):8798-803 PMID: 16723391
  77. DNA protein kinase-dependent G2 checkpoint revealed following knockdown of ataxia-telangiectasia mutated in human mammary epithelial cells.
    Cancer Res. 2008 Jan 1;68(1):89-97 PMID: 18172300
  78. Enhanced intra-switch region recombination during immunoglobulin class switch recombination in 53BP1-/- B cells.
    Eur J Immunol. 2007 Jan;37(1):235-9 PMID: 17183606
  79. The DNA-dependent protein kinase interacts with DNA to form a protein-DNA complex that is disrupted by phosphorylation.
    Biochemistry. 2002 Oct 22;41(42):12706-14 PMID: 12379113
  80. Mechanism and regulation of class switch recombination.
    Annu Rev Immunol. 2008;26:261-92 PMID: 18370922
  81. DNA-PKcs and Artemis function in the end-joining phase of immunoglobulin heavy chain class switch recombination.
    J Exp Med. 2008 Mar 17;205(3):557-64 PMID: 18316419
  82. Mechanism and control of V(D)J recombination versus class switch recombination: similarities and differences.
    Adv Immunol. 2005;86:43-112 PMID: 15705419
  83. Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessive form of the Hyper-IgM syndrome (HIGM2).
    Cell. 2000 Sep 1;102(5):565-75 PMID: 11007475
  84. Ataxia-telangiectasia mutated (ATM)-dependent activation of ATR occurs through phosphorylation of TopBP1 by ATM.
    J Biol Chem. 2007 Jun 15;282(24):17501-6 PMID: 17446169
  85. A pathway of double-strand break rejoining dependent upon ATM, Artemis, and proteins locating to gamma-H2AX foci.
    Mol Cell. 2004 Dec 3;16(5):715-24 PMID: 15574327
  86. Chk2-deficient mice exhibit radioresistance and defective p53-mediated transcription.
    EMBO J. 2002 Oct 1;21(19):5195-205 PMID: 12356735
  87. V(D)J recombination activates a p53-dependent DNA damage checkpoint in scid lymphocyte precursors.
    Genes Dev. 1996 Aug 15;10(16):2038-54 PMID: 8769647
Article Info
Journal
Molecular cell
Abbr.
Mol Cell
ISSN
1097-4164
DOI
10.1016/j.molcel.2009.04.025
Published
2009-05-15
Pages
285-97
Language
English
Region
United States
NLM ID
9802571
PMCID
PMC2709792
Subset
IM
Grants
Intramural NIH HHS · Z01 BC010283-10 · United States
Intramural NIH HHS · Z99 CA999999 · United States
Howard Hughes Medical Institute · United States
External Links
PubMed source DOI: 10.1016/j.molcel.2009.04.025 PMC Full Text View journal details
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