Making Ends Meet:

1

• Making Ends Meet
• This thing called Ku

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Ku

• First discovered as autoantigen in PM/Scl
  patients
• Name derived from original patients 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

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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

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Ku70, Ku80, and DNA-PKcs associate to form DNA-PK
Featherstone, C., and Jackson, S. Mutat Res. 1999
May 14434(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

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NHEJ proteins in S. cerevisiae and human cells
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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

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• 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

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

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• 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

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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

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• 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

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DNA-PK in NHEJ
Featherstone, C., and Jackson, S. Mutat Res. 1999
May 14434(1)3-15. Review.
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Ku is implicatedin transcription

• 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

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• 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

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Physiologicalfunctions of Ku

• 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

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Yeast Ku intelomere maintenance

• 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

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• 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 14434(1)3-15. Review.