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.
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).
Cheung
Joyce C Y
JC
Department of Biochemistry and Molecular Biology, Johns Hopkins University, Bloomberg School of Public Health, Baltimore, MD 21205, USA.
Salerno
Brenda
B
Hanakahi
Les A
LA
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
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
J Cell Physiol. 2007 Nov;213(2):475-83
17492771
Nat Cell Biol. 2006 Jul;8(7):668-76
16783364
DNA Repair (Amst). 2006 Sep 8;5(9-10):1021-9
16807135
DNA Repair (Amst). 2004 Aug-Sep;3(8-9):817-26
15279766
Science. 2005 Sep 2;309(5740):1534-9
16141067
Nature. 2007 Sep 27;449(7161):478-82
17713479
Science. 1999 Jul 2;285(5424):96-100
10390371
Cell. 2000 Sep 15;102(6):721-9
11030616
Cell Signal. 2001 Mar;13(3):151-8
11282453
Science. 2003 Jan 3;299(5603):112-4
12434013
Biochemistry. 2001 Aug 14;40(32):9638-46
11583164
Oncogene. 2005 Feb 3;24(6):949-61
15592499
Nature. 2007 Sep 27;449(7161):483-6
17898768
EMBO J. 2002 Apr 15;21(8):2038-44
11953323
Proc Natl Acad Sci U S A. 2007 Oct 2;104(40):15653-8
17895383
Genes Dev. 1999 Apr 15;13(8):916-34
10215620
Mol Cell Biol. 2003 Aug;23(16):5836-48
12897153
Nucleic Acids Res. 1999 Sep 1;27(17):3494-502
10446239
Cell. 2006 Jan 27;124(2):287-99
16439204
J Biol Chem. 2002 Mar 29;277(13):10756-9
11821378
Nucleic Acids Res. 1983 Mar 11;11(5):1475-89
6828386
Mol Cell Biol. 1990 Dec;10(12):6460-71
2247066
Nat Cell Biol. 2007 Aug;9(8):978-81
17643113
DNA Repair (Amst). 2005 Jul 12;4(7):749-59
15978533
DNA Repair (Amst). 2002 Mar 28;1(3):225-35
12509254
Nature. 2001 Aug 9;412(6847):607-14
11493912
Proc Natl Acad Sci U S A. 2006 Dec 5;103(49):18597-602
17124166
Proc Natl Acad Sci U S A. 1998 Nov 24;95(24):14066-70
9826654
Mol Cell Biol. 1990 Dec;10(12):6472-81
2247067
J Biol Chem. 2007 Apr 6;282(14):10605-13
17289670
DNA Repair (Amst). 2007 Jun 1;6(6):712-22
17241822
J Biol Chem. 2002 Mar 22;277(12):9741-8
11796732
Cell. 2006 Jan 27;124(2):301-13
16439205
Nature. 2007 Apr 5;446(7136):640-5
17410169
Int J Radiat Oncol Biol Phys. 2007 Aug 1;68(5):1462-70
17674976
Adv Enzyme Regul. 2001;41:57-71
11384737
Proc Natl Acad Sci U S A. 2005 Feb 8;102(6):1877-82
15671175
Genes Dev. 2002 Sep 15;16(18):2333-8
12231622
J Biol Chem. 2005 Feb 11;280(6):4264-9
15561716
Protein Expr Purif. 2007 Mar;52(1):139-45
17110127