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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
Recently shown to also bind:
ss gaps
ss bubbles
5’ or 3’ overhangs
hairpin ends
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
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.
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
NHEJ proteins in S.
cerevisiae and human cells
Human
Yeast
Properties
KU70
KU80 (KU86)
yKu70p (Hdf1p)
yKu80p (Hdf2p)
 Heterodimer comprises DNA-end-binding
component of DNA-PK
 Required for efficient NHEJ
 Confers catalytic function to DNA-PK complex
 No clear yeast homologue
 XRCC4 stimulates DNA ligase IV activity in
vitro
 Lif1p stabilizes Lig4p
 DNA end-joining activity
 Yeast homologue functions in Ku-dependent
NHEJ pathway
 Interacts with Mre11
 Homologous to SbcC exonuclease in E. Coli
 3’-5’ exonuclease & hairpin endonuclease
activity
 Homologous to SbcD exonuclease in E. Coli
DNA-PKcs
XRCC4
Lif1p
DNA ligase IV
Lig4p (Dnl4p)
RAD50
Rad50p
MRE11
Mre11p
Linking Ku with
DNA 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
SCID (severe combined immuno-deficiency)
mice
radiosensitive, defective in DSB repair
characteristic of a DNA-PKcs defect
radiosensitivity complemented by XRCC7 (DNAPKcs) gene
immunodeficiency due to V(D)J defect
• cells cannot properly rearrange immunoglobulin and Tcell receptor gene segments
• cannot maturate and diversify antibodies and T-cell
receptors
• Ku70 or Ku80 knock-outs have immuno-deficiency
phenotype similar to SCID
All components of DNA-PK function in
generating diverse antigen-binding
functions of mammalian immune system
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
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
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
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
Ku can translocate along DNA in ATPindependent 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
DNA-PK in NHEJ
Featherstone, C., and Jackson, S. Mutat Res. 1999 May 14;434(1):3-15. Review.
Ku is implicated
in 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
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
Physiological
functions 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
Yeast Ku in
telomere 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
 Ku clusters yeast
telomeres to peripheral
sites in nucleus
Featherstone, C., and Jackson, S. Mutat Res. 1999 May 14;434(1):3-15. Review.
In diploids, telomeres
usually found in 6-7
clusters around nuclear
periphery
In Ku subunit mutants,
more clusters in random
locations