Specific interaction of IP6 with human Ku70/80, the DNA‐binding subunit of DNA‐PK

In eukaryotic cells, DNA double‐strand breaks can be repaired by non‐homologous end‐joining, a process dependent upon Ku70/80, XRCC4 and DNA ligase IV. In mammals, this process also requires DNA‐PKcs, the catalytic subunit of the DNA‐dependent protein kinase DNA‐PK. Previously, inositol hexakisphosphate (IP6) was shown to be bound by DNA‐PK and to stimulate DNA‐PK‐dependent end‐joining in vitro. Here, we localize IP6 binding to the Ku70/80 subunits of DNA‐ PK, and show that DNA‐PKcs alone exhibits no detectable affinity for IP6. Moreover, proteolysis mapping of Ku70/80 in the presence and absence of IP6 indicates that binding alters the conformation of the Ku70/80 heterodimer. The yeast homologue of Ku70/80, yKu70/80, fails to bind IP6, indicating that the function of IP6 in non‐homologous end‐joining, like that of DNA‐PKcs, is unique to the mammalian end‐joining process.

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

[2]  J. Kearney,et al.  Ku80 is required for addition of N nucleotides to V(D)J recombination junctions by terminal deoxynucleotidyl transferase. , 2001, Nucleic acids research.

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

[4]  Lucio Comai,et al.  Functional Interaction between Ku and the Werner Syndrome Protein in DNA End Processing* , 2000, The Journal of Biological Chemistry.

[5]  S. West,et al.  Binding of Inositol Phosphate to DNA-PK and Stimulation of Double-Strand Break Repair , 2000, Cell.

[6]  S. Gasser.  A sense of the end. , 2000, Science.

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

[8]  J. Hoeijmakers,et al.  Homologous and non‐homologous recombination differentially affect DNA damage repair in mice , 2000, The EMBO journal.

[9]  B. Lehnert,et al.  DNA double-strand break repair proteins are required to cap the ends of mammalian chromosomes. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[10]  E. Blackburn,et al.  Ku is associated with the telomere in mammals. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[11]  S. Jackson,et al.  Mapping of protein-protein interactions within the DNA-dependent protein kinase complex. , 1999, Nucleic acids research.

[12]  P. Jeggo,et al.  The C Terminus of Ku80 Activates the DNA-Dependent Protein Kinase Catalytic Subunit , 1999, Molecular and Cellular Biology.

[13]  M. Gellert,et al.  DNA binding of Xrcc4 protein is associated with V(D)J recombination but not with stimulation of DNA ligase IV activity , 1999, The EMBO journal.

[14]  D. Chan,et al.  DNA-dependent protein kinase phosphorylation sites in Ku 70/80 heterodimer. , 1999, Biochemistry.

[15]  F. Alt,et al.  A Critical Role for DNA End-Joining Proteins in Both Lymphogenesis and Neurogenesis , 1998, Cell.

[16]  M. Falasca,et al.  Specificity and Promiscuity in Phosphoinositide Binding by Pleckstrin Homology Domains* , 1998, The Journal of Biological Chemistry.

[17]  F. Alt,et al.  Late embryonic lethality and impaired V (D)J recombination in mice lacking DNA ligase IV , 1998, Nature.

[18]  K. Schwarz,et al.  DNA ligase IV is essential for V(D)J recombination and DNA double-strand break repair in human precursor lymphocytes. , 1998, Molecular cell.

[19]  R. West,et al.  Productive and Nonproductive Complexes of Ku and DNA-Dependent Protein Kinase at DNA Termini , 1998, Molecular and Cellular Biology.

[20]  S. Jackson,et al.  DNA end-joining: from yeast to man. , 1998, Trends in biochemical sciences.

[21]  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.

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

[23]  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.

[24]  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.

[25]  G. Chu.  Double Strand Break Repair* , 1997, The Journal of Biological Chemistry.

[26]  S. Jackson,et al.  Mammalian DNA double-strand break repair protein XRCC4 interacts with DNA ligase IV , 1997, Current Biology.

[27]  M. Lieber,et al.  Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells , 1997, Nature.

[28]  M. Lieber,et al.  Protein-protein and protein-DNA interaction regions within the DNA end-binding protein Ku70-Ku86 , 1996, Molecular and cellular biology.

[29]  D. Chan,et al.  The DNA-dependent Protein Kinase Is Inactivated by Autophosphorylation of the Catalytic Subunit (*) , 1996, The Journal of Biological Chemistry.

[30]  D. Weaver,et al.  Regulation and repair of double-strand DNA breaks. , 1996, Critical reviews in eukaryotic gene expression.

[31]  T. Hunter,et al.  When is a lipid kinase not a lipid kinase? When it is a protein kinase , 1995, Cell.

[32]  P. Jeggo,et al.  DNA double-strand break repair and V(D)J recombination: involvement of DNA-PK. , 1995, Trends in biochemical sciences.

[33]  A. Dvir,et al.  Purification and characterization of a template-associated protein kinase that phosphorylates RNA polymerase II. , 1993, The Journal of biological chemistry.

[34]  H. Busch,et al.  A 10S particle released from deoxyribonuclease-sensitive regions of HeLa cell nuclei contains the 86-kilodalton-70-kilodalton protein complex. , 1986, Biochemistry.