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

• Published in: Nucleic acids research Vol. 36; no. 17; pp. 5713 - 5726
• Main Authors: Cheung, Joyce C.Y, Salerno, Brenda, Hanakahi, Les A
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.10.2008; Oxford Publishing Limited (England)
• Subjects: Antigens, Nuclear - chemistry; Antigens, Nuclear - genetics; Antigens, Nuclear - physiology; Binding Sites; DNA Repair; DNA-Activated Protein Kinase - metabolism; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; DNA-Binding Proteins - physiology; HeLa Cells; Humans; Ku Autoantigen; Mutation; Nucleic Acid Enzymes; Phytic Acid - chemistry; Phytic Acid - metabolism; Protein Binding; Protein Subunits - metabolism
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkn572

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 (IP₆) was previously found to stimulate NHEJ in vitro and Ku was identified as an IP₆-binding factor. Through mutational analysis, we identified a bipartite IP₆-binding site in Ku and generated IP₆-binding mutants that ranged from 1.22% to 58.48% of wild-type binding. Significantly, these Ku IP₆-binding mutants were impaired for participation in NHEJ in vitro and we observed a positive correlation between IP₆ binding and NHEJ. Ku IP₆-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 IP₆-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 IP₆.