title: Evidence for an inositol hexakisphosphate-dependent role for Ku in mammalian nonhomologous end joining that is independent of its role in the DNA-dependent protein kinase.
abstract: Nonhomologous end-joining (NHEJ) is an important pathway for the repair of DNA double-strand breaks (DSBs) and plays a critical role in maintaining genomic stability in mammalian cells. While Ku70/80 (Ku) functions in NHEJ as part of the DNA-dependent protein kinase (DNA-PK), genetic evidence indicates that the role of Ku in NHEJ goes beyond its participation in DNA-PK. Inositol hexakisphosphate (IP(6)) was previously found to stimulate NHEJ in vitro and Ku was identified as an IP(6)-binding factor. Through mutational analysis, we identified a bipartite IP(6)-binding site in Ku and generated IP(6)-binding mutants that ranged from 1.22% to 58.48% of wild-type binding. Significantly, these Ku IP(6)-binding mutants were impaired for participation in NHEJ in vitro and we observed a positive correlation between IP(6) binding and NHEJ. Ku IP(6)-binding mutants were separation-of-function mutants that bound DNA and activated DNA-PK as well as wild-type Ku. Our observations identify a hitherto undefined IP(6)-binding site in Ku and show that this interaction is important for DSB repair by NHEJ in vitro. Moreover, these data indicate that in addition to binding of exposed DNA termini and activation of DNA-PK, the Ku heterodimer plays a role in mammalian NHEJ that is regulated by binding of IP(6).
authors.affiliation: Department of Biochemistry and Molecular Biology, Johns Hopkins University, Bloomberg School of Public Health, Baltimore, MD 21205, USA.
authors.forename: Joyce C Y
authors.initials: JC
authors.lastname: Cheung
authors.forename: Brenda
authors.initials: B
authors.lastname: Salerno
authors.forename: Les A
authors.initials: LA
authors.lastname: Hanakahi
mesh_terms: D034961:Antigens, Nuclear / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D001665:Binding Sites; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D000072200:Ku Autoantigen; D009154:Mutation; D010833:Phytic Acid / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism
chemical_list: D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D021122:Protein Subunits; D010833:Phytic Acid; D051747:DNA-Activated Protein Kinase; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen
references.citation: J Cell Physiol. 2007 Nov;213(2):475-83
references.pmid: 17492771
references.citation: Nat Cell Biol. 2006 Jul;8(7):668-76
references.pmid: 16783364
references.citation: DNA Repair (Amst). 2006 Sep 8;5(9-10):1021-9
references.pmid: 16807135
references.citation: DNA Repair (Amst). 2004 Aug-Sep;3(8-9):817-26
references.pmid: 15279766
references.citation: Science. 2005 Sep 2;309(5740):1534-9
references.pmid: 16141067
references.citation: Nature. 2007 Sep 27;449(7161):478-82
references.pmid: 17713479
references.citation: Science. 1999 Jul 2;285(5424):96-100
references.pmid: 10390371
references.citation: Cell. 2000 Sep 15;102(6):721-9
references.pmid: 11030616
references.citation: Cell Signal. 2001 Mar;13(3):151-8
references.pmid: 11282453
references.citation: Science. 2003 Jan 3;299(5603):112-4
references.pmid: 12434013
references.citation: Biochemistry. 2001 Aug 14;40(32):9638-46
references.pmid: 11583164
references.citation: Oncogene. 2005 Feb 3;24(6):949-61
references.pmid: 15592499
references.citation: Nature. 2007 Sep 27;449(7161):483-6
references.pmid: 17898768
references.citation: EMBO J. 2002 Apr 15;21(8):2038-44
references.pmid: 11953323
references.citation: Proc Natl Acad Sci U S A. 2007 Oct 2;104(40):15653-8
references.pmid: 17895383
references.citation: Genes Dev. 1999 Apr 15;13(8):916-34
references.pmid: 10215620
references.citation: Mol Cell Biol. 2003 Aug;23(16):5836-48
references.pmid: 12897153
references.citation: Nucleic Acids Res. 1999 Sep 1;27(17):3494-502
references.pmid: 10446239
references.citation: Cell. 2006 Jan 27;124(2):287-99
references.pmid: 16439204
references.citation: J Biol Chem. 2002 Mar 29;277(13):10756-9
references.pmid: 11821378
references.citation: Nucleic Acids Res. 1983 Mar 11;11(5):1475-89
references.pmid: 6828386
references.citation: Mol Cell Biol. 1990 Dec;10(12):6460-71
references.pmid: 2247066
references.citation: Nat Cell Biol. 2007 Aug;9(8):978-81
references.pmid: 17643113
references.citation: DNA Repair (Amst). 2005 Jul 12;4(7):749-59
references.pmid: 15978533
references.citation: DNA Repair (Amst). 2002 Mar 28;1(3):225-35
references.pmid: 12509254
references.citation: Nature. 2001 Aug 9;412(6847):607-14
references.pmid: 11493912
references.citation: Proc Natl Acad Sci U S A. 2006 Dec 5;103(49):18597-602
references.pmid: 17124166
references.citation: Proc Natl Acad Sci U S A. 1998 Nov 24;95(24):14066-70
references.pmid: 9826654
references.citation: Mol Cell Biol. 1990 Dec;10(12):6472-81
references.pmid: 2247067
references.citation: J Biol Chem. 2007 Apr 6;282(14):10605-13
references.pmid: 17289670
references.citation: DNA Repair (Amst). 2007 Jun 1;6(6):712-22
references.pmid: 17241822
references.citation: J Biol Chem. 2002 Mar 22;277(12):9741-8
references.pmid: 11796732
references.citation: Cell. 2006 Jan 27;124(2):301-13
references.pmid: 16439205
references.citation: Nature. 2007 Apr 5;446(7136):640-5
references.pmid: 17410169
references.citation: Int J Radiat Oncol Biol Phys. 2007 Aug 1;68(5):1462-70
references.pmid: 17674976
references.citation: Adv Enzyme Regul. 2001;41:57-71
references.pmid: 11384737
references.citation: Proc Natl Acad Sci U S A. 2005 Feb 8;102(6):1877-82
references.pmid: 15671175
references.citation: Genes Dev. 2002 Sep 15;16(18):2333-8
references.pmid: 12231622
references.citation: J Biol Chem. 2005 Feb 11;280(6):4264-9
references.pmid: 15561716
references.citation: Protein Expr Purif. 2007 Mar;52(1):139-45
references.pmid: 17110127