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Making Ends Meet: This thing called Ku

Making Ends Meet: This thing called Ku. Ku. First discovered as autoantigen in PM/Scl patients Name derived from original patient’s name Antibodies against Ku also found in patients with other autoimmune diseases. Purified protein binds tightly to free ends of linear dsDNA

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Making Ends Meet: This thing called Ku

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  1. Making Ends Meet: This thing called Ku

  2. Ku • First discovered as autoantigen in PM/Scl patients • Name derived from original patient’s name • Antibodies against Ku also found in patients with other autoimmune diseases

  3. Purified protein binds tightly to free ends of linear dsDNA • Recently shown to also bind: • ss gaps • ss bubbles • 5’ or 3’ overhangs • hairpin ends

  4. Human Ku • Heterodimer • Ku70 (69 kDa) • Ku80 (83 kDa) • Conserved across species by size only, not amino acid sequence • Might act as dimer of dimers

  5. Ku70, Ku80, and DNA-PKcs associate to form DNA-PK Featherstone, C., and Jackson, S. Mutat Res. 1999 May 14;434(1):3-15. Review.

  6. Ku and DNA-PKcs can repair damage caused by • physiological oxidative reactions, • V(D)J recombination, • certain drugs, and • ionizing radiation-induced DNA DSBs • Ku knock-out mice and yeast reveal additional functions for Ku apart from DNA repair • maintenance of genomic integrity

  7. NHEJ proteins in S. cerevisiae and human cells

  8. Linking Ku withDNA DSB repair • In mammalian systems • 1994 - DNA-PKcs- & Ku80-deficient cells have defective DNA DSB rejoining • extreme sensitivity to ionizing radiation and other agents that cause DNA DSBs • less sensitive to UV, alkylating agents, mitomycin C • Ku70 knock-out phenotype • hypersensitive to ionizing radiation • defective DNA-end binding activity due to Ku • cannot support V(D)J recombination

  9. SCID (severe combined immuno-deficiency) mice • radiosensitive, defective in DSB repair characteristic of a DNA-PKcs defect • radiosensitivity complemented by XRCC7 (DNA-PKcs) gene • immunodeficiency due to V(D)J defect • cells cannot properly rearrange immunoglobulin and T-cell receptor gene segments • cannot maturate and diversify antibodies and T-cell receptors • Ku70 or Ku80 knock-outs have immuno-deficiency phenotype similar to SCID

  10. All components of DNA-PK function in generating diverse antigen-binding functions of mammalian immune system

  11. In cerevisiae • Heterodimer functions in NHEJ • ligates two DNA ends without extensive homology • little or no nucleotide loss • Although NHEJ repairs most vertebrate DSBs, in yeast repaired mainly by homologous recombination • NHEJ important in haploid G1 • no homologous chromosomes present for homologous recombination

  12. Impair yKu70p or yKu80p, severely impair NHEJ • But no obvious DNA-PKcs homologue • functions mediated by DNA-PKcs do not occur in yeast • mediated by other polypeptides • Mec1p, Tel1p

  13. How does Ku function in DNA DSB repair? • Ku binds tightly and rapidly to DNA ends • likely Ku can recognize various broken DNA structures in cells • might prevent exonuclease activity on DNA • but V(D)J intermediates stable without Ku • possibility: Ku holds two DNA ends on both sides of DSB • facilitates processing and ligation by other repair components

  14. Can Ku function in targeting nucleases (Rad50p, Mre11p) to DSB site and/or modulate nuclease activities? • SbcC, SbcD act as nucleases in E. Coli • RAD50, MRE11, XRS2 form epistasis group required for NHEJ in yeast

  15. Ku can translocate along DNA in ATP-independent fashion • each dimer binds to DNA end • slides apart from each other to open helix • Ku has weakly processive DNA helicase activity • ends presented with regions of microhomology • ends anneal together

  16. DNA-PK in NHEJ Featherstone, C., and Jackson, S. Mutat Res. 1999 May 14;434(1):3-15. Review.

  17. DNA-PK phosphorylates transcription factors and regulatory C-terminal domain of RNA polymerase II in vitro no evidence yet that transcriptional proteins act as substrates for Ku in vivo Ku binds sequences in transcriptional regulatory elements no clear consensus sequence for Ku DNA-binding Ku is implicatedin transcription

  18. DNA-PK can phosphorylate RNA polymerase I transcription apparatus • responsible for transcription of large ribosomal RNA precursor • Ku binding changes local conformation of DNA substrate • equilibrium shifts from euchromatin to heterochromatin • might repress transcription • might facilitate juxtaposition of DNA ends

  19. Ku70, Ku80 knockouts in mice have similar phenotype to SCID V(D)J defects arrest lymphocyte development Ku70, Ku80 -/- mice are runts compared to +/- littermates Number of cell divisions in development limited by impaired ability to repair endogenously generated DNA damage Ku-deficient cells might take longer to repair this damage Ku80 -/- dams fail to nurture their pups Physiologicalfunctions of Ku

  20. Disruption of yKu70p and yKu80p genes affect telomeric silencing and telomere length maintenance inactivate Ku, lose telomeric silencing inactivate Ku, shorten telomeres Model: Ku binds double-stranded telomeric ends, blocks accessibility of certain nucleases in most of cell cycle. Ku displaced from telomeric ends during S phase, allowing exonucleolytic degradation of one strand, creating ssDNA binding site for telomerase Yeast Ku intelomere maintenance

  21. Ku clusters yeast telomeres to peripheral sites in nucleus • In diploids, telomeres usually found in 6-7 clusters around nuclear periphery • In Ku subunit mutants, more clusters in random locations Featherstone, C., and Jackson, S. Mutat Res. 1999 May 14;434(1):3-15. Review.

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