Biochemical evidence for Ku-independent backup pathways of NHEJ.

Cells of higher eukaryotes process within minutes double strand breaks (DSBs) in their genome using a non-homologous end joining (NHEJ) apparatus that engages DNA-PKcs, Ku, DNA ligase IV, XRCC4 and other as of yet unidentified factors. Although chemical inhibition, or mutation, in any of these factors delays processing, cells ultimately remove the majority of DNA DSBs using an alternative pathway operating with an order of magnitude slower kinetics. This alternative pathway is active in mutants deficient in genes of the RAD52 epistasis group and frequently joins incorrect ends. We proposed, therefore, that it reflects an alternative form of NHEJ that operates as a backup (B-NHEJ) to the DNA-PK-dependent (D-NHEJ) pathway, rather than homology directed repair of DSBs. The present study investigates the role of Ku in the coordination of these pathways using as a model end joining of restriction endonuclease linearized plasmid DNA in whole cell extracts. Efficient, error-free, end joining observed in such in vitro reactions is strongly inhibited by anti-Ku antibodies. The inhibition requires DNA-PKcs, despite the fact that Ku efficiently binds DNA ends in the presence of antibodies, or in the absence of DNA-PKcs. Strong inhibition of DNA end joining is also mediated by wortmannin, an inhibitor of DNA-PKcs, in the presence but not in the absence of Ku, and this inhibition can be rescued by pre-incubating the reaction with double stranded oligonucleotides. The results are compatible with a role of Ku in directing end joining to a DNA-PK dependent pathway, mediated by efficient end binding and productive interactions with DNA-PKcs. On the other hand, efficient end joining is observed in extracts of cells lacking DNA-PKcs, as well as in Ku-depleted extracts in line with the operation of alternative pathways. Extracts depleted of Ku and DNA-PKcs rejoin blunt ends, as well as homologous ends with 3' or 5' protruding single strands with similar efficiency, but addition of Ku suppresses joining of blunt ends and homologous ends with 3' overhangs. We propose that the affinity of Ku for DNA ends, particularly when cooperating with DNA-PKcs, suppresses B-NHEJ by quickly and efficiently binding DNA ends and directing them to D-NHEJ for rapid joining. A chromatin-based model of DNA DSB rejoining accommodating biochemical and genetic results is presented and deviations between in vitro and in vivo results discussed.

[1]  M. Jung,et al.  Ku proteins join DNA fragments as shown by atomic force microscopy. , 1997, Cancer research.

[2]  W. Dynan,et al.  Reconstitution of the mammalian DNA double-strand break end-joining reaction reveals a requirement for an Mre11/Rad50/NBS1-containing fraction. , 2002, Nucleic acids research.

[3]  J. Haber DNA repair: Gatekeepers of recombination , 1999, Nature.

[4]  T. M. Rünger,et al.  Reduced joining of DNA double strand breaks with an abnormal mutation spectrum in rodent mutants of DNA-PKcs and Ku80. , 1998, International journal of radiation biology.

[5]  D. Ramsden,et al.  Ku Recruits the XRCC4-Ligase IV Complex to DNA Ends , 2000, Molecular and Cellular Biology.

[6]  K. Sakaguchi,et al.  Human DNA-activated protein kinase phosphorylates serines 15 and 37 in the amino-terminal transactivation domain of human p53 , 1992, Molecular and cellular biology.

[7]  H. Lu,et al.  Ku autoantigen is the regulatory component of a template-associated protein kinase that phosphorylates RNA polymerase II. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[8]  L. Povirk,et al.  End-joining of Free Radical-mediated DNA Double-strand Breaks in Vitro Is Blocked by the Kinase Inhibitor Wortmannin at a Step Preceding Removal of Damaged 3′ Termini* , 1996, The Journal of Biological Chemistry.

[9]  Huichen Wang,et al.  Genetic evidence for the involvement of DNA ligase IV in the DNA-PK-dependent pathway of non-homologous end joining in mammalian cells. , 2001, Nucleic acids research.

[10]  L. Povirk,et al.  Accurate in Vitro End Joining of a DNA Double Strand Break with Partially Cohesive 3′-Overhangs and 3′-Phosphoglycolate Termini , 2001, The Journal of Biological Chemistry.

[11]  P. Baumann,et al.  DNA end-joining catalyzed by human cell-free extracts. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[12]  A. Griffith,et al.  Binding of Ku protein to DNA. Measurement of affinity for ends and demonstration of binding to nicks. , 1993, The Journal of biological chemistry.

[13]  R. Strick,et al.  Cation–chromatin binding as shown by ion microscopy is essential for the structural integrity of chromosomes , 2001, The Journal of cell biology.

[14]  F. Alt,et al.  Interplay of p53 and DNA-repair protein XRCC4 in tumorigenesis, genomic stability and development , 2000, Nature.

[15]  S. Jackson,et al.  Sensing and repairing DNA double-strand breaks. , 2002, Carcinogenesis.

[16]  D. Roth,et al.  Mechanisms of nonhomologous recombination in mammalian cells , 1985, Molecular and cellular biology.

[17]  J. Sarkaria,et al.  Inhibition of phosphoinositide 3-kinase related kinases by the radiosensitizing agent wortmannin. , 1998, Cancer research.

[18]  David J. Chen,et al.  The DNA-dependent Protein Kinase Catalytic Activity Regulates DNA End Processing by Means of Ku Entry into DNA* , 1999, The Journal of Biological Chemistry.

[19]  Elke Feldmann,et al.  DNA double-strand break repair in cell-free extracts from Ku80-deficient cells: implications for Ku serving as an alignment factor in non-homologous DNA end joining , 2000, Nucleic Acids Res..

[20]  T. Lange Telomere Capping--One Strand Fits All , 2001 .

[21]  Kyung-Jong Lee,et al.  DNA Ligase IV and XRCC4 Form a Stable Mixed Tetramer That Functions Synergistically with Other Repair Factors in a Cell-free End-joining System* , 2000, The Journal of Biological Chemistry.

[22]  T. de Lange Cell biology. Telomere capping--one strand fits all. , 2001, Science.

[23]  T. Petes,et al.  The DNA-binding protein Hdf1p (a putative Ku homologue) is required for maintaining normal telomere length in Saccharomyces cerevisiae. , 1996, Nucleic acids research.

[24]  S. Jackson,et al.  The DNA-dependent protein kinase: Requirement for DNA ends and association with Ku antigen , 1993, Cell.

[25]  J. Hoeijmakers,et al.  Different types of V(D)J recombination and end‐joining defects in DNA double‐strand break repair mutant mammalian cells , 2002, European journal of immunology.

[26]  G. Iliakis,et al.  Homologous recombination as a potential target for caffeine radiosensitization in mammalian cells: reduced caffeine radiosensitization in XRCC2 and XRCC3 mutants , 2000, Oncogene.

[27]  W. Dynan,et al.  Interaction of Ku protein and DNA-dependent protein kinase catalytic subunit with nucleic acids. , 1998, Nucleic acids research.

[28]  O. Hammarsten,et al.  DNA-dependent protein kinase: DNA binding and activation in the absence of Ku. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[29]  H. Koyama,et al.  DNA ligase IV-deficient cells are more resistant to ionizing radiation in the absence of Ku70: Implications for DNA double-strand break repair , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[30]  Y. Yamaguchi-Iwai,et al.  Homologous recombination and non‐homologous end‐joining pathways of DNA double‐strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells , 1998, The EMBO journal.

[31]  J. Dodge,et al.  Wortmannin, a potent and selective inhibitor of phosphatidylinositol-3-kinase. , 1994, Cancer research.

[32]  L. Povirk,et al.  Implication of DNA-dependent protein kinase in an early, essential, local phosphorylation event during end-joining of DNA double-strand breaks in vitro. , 1998, Biochemistry.

[33]  D. Roth,et al.  Nonhomologous recombination in mammalian cells: role for short sequence homologies in the joining reaction , 1986, Molecular and cellular biology.

[34]  D. Baltimore,et al.  In vitro V(D)J recombination: signal joint formation. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[35]  S. Jackson,et al.  DNA-dependent protein kinase. , 1997, The international journal of biochemistry & cell biology.

[36]  M. Löbrich,et al.  Repair of x-ray-induced DNA double-strand breaks in specific Not I restriction fragments in human fibroblasts: joining of correct and incorrect ends. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[37]  J. Brown,et al.  Characterization of the DNA double strand break repair defect in scid mice. , 1993, Cancer research.

[38]  F. Alt,et al.  Unrepaired DNA Breaks in p53-Deficient Cells Lead to Oncogenic Gene Amplification Subsequent to Translocations , 2002, Cell.

[39]  T. de Lange Protection of mammalian telomeres , 2002, Oncogene.

[40]  J. Walker,et al.  Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair , 2001, Nature.

[41]  R. Mirzayans,et al.  Isolation of two cell lines from a human malignant glioma specimen differing in sensitivity to radiation and chemotherapeutic drugs. , 1993, Radiation research.

[42]  M. Hande,et al.  Ku acts in a unique way at the mammalian telomere to prevent end joining. , 2000, Genes & development.

[43]  T. Lange Protection of mammalian telomeres , 2002, Oncogene.

[44]  D. Lane,et al.  Ku Selectively Transfers between DNA Molecules with Homologous Ends* , 1997, The Journal of Biological Chemistry.

[45]  B. Nevaldine,et al.  The scid defect results in much slower repair of DNA double-strand breaks but not high levels of residual breaks. , 1997, Radiation research.

[46]  D. Roth,et al.  Double-strand break repair in Ku86- and XRCC4-deficient cells. , 1998, Nucleic acids research.

[47]  M. Lieber,et al.  Analysis of the defect in DNA end joining in the murine scid mutation , 1992, Molecular and cellular biology.

[48]  George Iliakis,et al.  Efficient rejoining of radiation-induced DNA double-strand breaks in vertebrate cells deficient in genes of the RAD52 epistasis group , 2001, Oncogene.

[49]  M. Connelly,et al.  DNA-dependent protein kinase catalytic subunit: A relative of phosphatidylinositol 3-kinase and the ataxia telangiectasia gene product , 1995, Cell.

[50]  S. Galande,et al.  Poly(ADP-ribose) Polymerase and Ku Autoantigen Form a Complex and Synergistically Bind to Matrix Attachment Sequences* , 1999, The Journal of Biological Chemistry.

[51]  S. Jackson,et al.  DNA repair: How Ku makes ends meet , 2001, Current Biology.

[52]  M. Falzon,et al.  EBP-80, a transcription factor closely resembling the human autoantigen Ku, recognizes single- to double-strand transitions in DNA. , 1993, The Journal of biological chemistry.

[53]  S. Thode,et al.  A novel pathway of DNA end-to-end joining , 1990, Cell.

[54]  T Hyslop,et al.  DNA-dependent protein kinase stimulates an independently active, nonhomologous, end-joining apparatus. , 2000, Cancer research.

[55]  A. Arnberg,et al.  HeLa nuclear protein recognizing DNA termini and translocating on DNA forming a regular DNA-multimeric protein complex. , 1989, Journal of molecular biology.

[56]  D. Chan,et al.  Absence of p350 subunit of DNA-activated protein kinase from a radiosensitive human cell line , 1995, Science.

[57]  S. Jackson,et al.  Identification of a Saccharomyces cerevisiae Ku80 homologue: roles in DNA double strand break rejoining and in telomeric maintenance. , 1996, Nucleic acids research.

[58]  P. Labhart,et al.  Nonhomologous DNA end joining in cell-free systems. , 2001, European journal of biochemistry.

[59]  Thomas Ried,et al.  DNA repair protein Ku80 suppresses chromosomal aberrations and malignant transformation , 2000, Nature.

[60]  T. de Lange,et al.  Ku Binds Telomeric DNA in Vitro * , 1999, The Journal of Biological Chemistry.

[61]  Akira Shinohara,et al.  Rad51‐deficient vertebrate cells accumulate chromosomal breaks prior to cell death , 1998, The EMBO journal.

[62]  W. Zhang,et al.  On the mechanisms of Ku protein binding to DNA. , 1992, Biochemical and biophysical research communications.

[63]  D. Ramsden,et al.  Ku protein stimulates DNA end joining by mammalian DNA ligases: a direct role for Ku in repair of DNA double‐strand breaks , 1998, The EMBO journal.

[64]  P. Labhart Ku-Dependent Nonhomologous DNA End Joining inXenopus Egg Extracts , 1999, Molecular and Cellular Biology.

[65]  S. Jackson,et al.  Ku, a DNA repair protein with multiple cellular functions? , 1999, Mutation research.

[66]  D. Schild,et al.  Homologous recombinational repair of DNA ensures mammalian chromosome stability. , 2001, Mutation research.