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

KU70KU80 (KU86)

yKu70p (Hdf1p)yKu80p (Hdf2p)

Heterodimer comprises DNA-end-bindingcomponent of DNA-PK

Required for efficient NHEJDNA-PKcs

Confers catalytic function to DNA-PK complex No clear yeast homologue

XRCC4 Lif1p XRCC4 stimulates DNA ligase IV activity invitro

Lif1p stabilizes Lig4pDNA ligase IV Lig4p (Dnl4p) DNA end-joining activity

Yeast homologue functions in Ku-dependentNHEJ pathway

RAD50 Rad50p Interacts with Mre11 Homologous to SbcC exonuclease in E. Coli

MRE11 Mre11p 3’-5’ exonuclease & hairpin endonucleaseactivity

Homologous to SbcD exonuclease in E. Coli

Linking Ku withDNA DSB repair

In mammalian systems 1994 - DNA-PKcs- & Ku80-deficient cells have

defective DNA DSB rejoiningextreme sensitivity to ionizing radiation and other

agents that cause DNA DSBsless sensitive to UV, alkylating agents,

mitomycin C

Ku70 knock-out phenotypehypersensitive to ionizing radiationdefective DNA-end binding activity due to Kucannot support V(D)J recombination

SCID (severe combined immuno-deficiency) miceradiosensitive, defective in DSB repair characteristic of

a DNA-PKcs defectradiosensitivity complemented by XRCC7 (DNA-PKcs)

geneimmunodeficiency 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

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 homologylittle or no nucleotide loss

Although NHEJ repairs most vertebrate DSBs, in yeast repaired mainly by homologous recombination

NHEJ important in haploid G1no homologous chromosomes present for

homologous recombination

Impair yKu70p or yKu80p, severely impair NHEJ

But no obvious DNA-PKcs homologuefunctions mediated by DNA-PKcs do not

occur in yeastmediated 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 cellsmight prevent exonuclease activity on DNA

but V(D)J intermediates stable without Kupossibility: Ku holds two DNA ends on both

sides of DSBfacilitates 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 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

DNA-PK in NHEJ

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

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

DNA-PK can phosphorylate RNA polymerase I transcription apparatus responsible for transcription of large ribosomal

RNA precursorKu binding changes local conformation of

DNA substrate equilibrium shifts from euchromatin to

heterochromatin might repress transcription might facilitate juxtaposition of DNA ends

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

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

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.