Binding of Inositol Phosphate to DNA-PK and Stimulation of Double-Strand Break Repair

@article{Hanakahi2000BindingOI,
  title={Binding of Inositol Phosphate to DNA-PK and Stimulation of Double-Strand Break Repair},
  author={Les A. Hanakahi and Michael Bartlet-Jones and Claire Chappell and Darryl J. Pappin and Stephen C. West},
  journal={Cell},
  year={2000},
  volume={102},
  pages={721-729}
}
In mammalian cells, double-strand breaks in DNA can be repaired by nonhomologous end-joining (NHEJ), a process dependent upon Ku70/80, DNA-PKcs, XRCC4, and DNA ligase IV. Starting with HeLa cell-free extracts, which promote NHEJ in a reaction dependent upon all of these proteins, we have purified a novel factor that stimulates DNA end-joining in vitro. Using a combination of phosphorus NMR, mass spectroscopy, and strong anion exchange chromatography, we identify this factor as inositol… Expand
Binding of Inositol Hexakisphosphate (IP6) to Ku but Not to DNA-PKcs *
TLDR
It is shown that myo-inositol hexakisphosphate stimulates the joining of complementary DNA ends in a cell free system and that IP6 associates not with DNA-PKcs, but rather with Ku, the indispensable protein in the NHEJ pathway. Expand
Visualization of inositol phosphate-dependent mobility of Ku: depletion of the DNA-PK cofactor InsP6 inhibits Ku mobility.
TLDR
Results show that InsP 6 enhances Ku mobility through a discrete region of Ku70, and modulation of InsP6 levels in cells represents a potential avenue for regulating NHEJ by affecting the dynamics of Ku and hence its interaction with other nuclear proteins. Expand
Effect of the Inositol Polyphosphate InsP6 on DNA-PK–Dependent Phosphorylation
  • L. Hanakahi
  • Biology, Medicine
  • Molecular Cancer Research
  • 2011
TLDR
InsP6-dependent phosphorylation of two NHEJ factors, XRCC4 and XLF, in partially purified human cell extracts is described and binding of the inositol polyphosphate InsP6 by Ku70/80 may modulate the substrate specificity of the phosphoinositide-3-kinase–related protein kinase DNA-PK. Expand
Synthesis of Biotinylated Inositol Hexakisphosphate To Study DNA Double-Strand Break Repair and Affinity Capture of IP6-Binding Proteins.
TLDR
The synthesis, characterization, and application of biotin-labeled IP6 (IP6-biotin), which has biotin attached at position 2 of the myo-inositol ring via an aminohexyl linker, is reported on. Expand
The role of DNA dependent protein kinase in synapsis of DNA ends.
TLDR
A working model is presented in which DNA-PK creates a stable molecular bridge between two DNA ends that is remodeled after DNA- PK autophosphorylation in such a way that the extreme termini become accessible without disrupting synapsis. Expand
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
TLDR
Observations identify a hitherto undefined IP6-binding site in Ku and show that this interaction is important for DSB repair by NHEJ in vitro, indicating that in addition to binding of exposed DNA termini and activation of DNA-PK, the Ku heterodimer plays a role in mammalian N HEJ that is regulated by binding of IP6. Expand
Characterization of the macromolecular interactions of the key NHEJ components.
Non-homologous end-joining (NHEJ) is a major mechanism for repairing DNA double strand breaks in mammalian cells. Six ‘core’ NHEJ components have been identified to date: Ku70, Ku80, the DNAExpand
Specific interaction of IP6 with human Ku70/80, the DNA‐binding subunit of DNA‐PK
TLDR
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. Expand
Activation Mechanism of Protein Kinase B by DNA-dependent Protein Kinase Involved in the DNA Repair System
TLDR
It is proposed that DNA-PK has a potential role in insulin signaling as well as DNA-repair signaling pathway through its role in 2nd upstream kinase for protein kinase B activation. Expand
Structure of an activated DNA-PK and its implications for NHEJ.
TLDR
Cryoelectron microscopy structures of DNA-PKcs (DNA-PK catalytic subunit) bound to a DNA end or complexed with Ku70/80 and DNA in both inactive and activated forms reveal the sequential transition ofDNA- PK from inactive to activated forms. Expand
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