Cell polarity protein Par3 complexes with DNA-PK via Ku70 and regulates DNA double-strand break repair

The partitioning-defective 3 (Par3),a key component in the conserved Par3/Par6/aPKC complex,plays fundamentalroles in cell polarity.Herein we report the identification of Ku70 and Ku80 as novel Par3-interacting proteins throughan in vitro binding assay followed by liquid chromatography-tandem mass spectrometry.Ku70/Ku80 proteins are twokey regulatory subunits of the DNA-dependent protein kinase (DNA-PK),which plays an essential role in repairingdouble-strand DNA breaks (DSBs).We determined that the nuclear association of Par3 with Ku70/KuS0 was enhancedby y-irradiation (IR),a potent DSB inducer.Furthermore,DNA-PKcs,the catalytic subunit of DNA-PK,interacted withthe Par3/Ku70/Ku80 complex in response to IR.Par3 over-expression or knockdown was capable of up-or downregulat-ing DNA-PK activity,respectively.Moreover,the Par3 knockdown cells were found to be defective in random plasmidintegration,defective in DSB repair following IR,and radiosensitive,phenotypes similar to that of Ku70 knockdowncells.These findings identify Par3 as a novel component of the DNA-PK complex and implicate an unexpected link ofcell polarity to DSB repair.     

Interaction of human Ku70 with TRF2

Ku, a heterodimer of 70- and 80-kDa subunits, plays a general role in the metabolism of DNA ends in eukaryotic cells, including double-strand DNA break repair, V(D)J recombination, and maintenance of telomeres. We have utilized the yeast two-hybrid system to identify Ku70-interacting proteins other than Ku80. Two reactive clones were found to encode the dimerization domain of TRF2, a mammalian telomeric protein that binds to duplex TTAGGG repeats at chromosome ends. This interaction was confirmed using bacterial fusion proteins and co-immunoprecipitations from eukaryotic cells overexpressing TRF2. The transfected TFR2 colocalized with Ku70.     

β-Carotene-induced apoptosis is mediated with loss of Ku proteins in gastric cancer AGS cells

High dietary intakes and high blood levels of β-carotene are associated with a decreased incidence of various cancers. The anticancer effect of β-carotene is related to its pro-oxidant activity. DNA repair Ku proteins, as a heterodimer of Ku70 and Ku80, play a crucial role in DNA double-strand break repair. Reductions in Ku70/80 contribute to apoptosis. Previously, we showed that reactive oxygen species (ROS) activate caspase-3 which induces degradation of Ku proteins. In the present study, we investigated the mechanism of β-carotene-induced apoptosis of gastric cancer AGS cells by determining cell viability, DNA fragmentation, apoptotic indices (increases in cytochrome c and Bax, decrease in Bcl-2), ROS levels, mitochondrial membrane potential, caspase-3 activity, Ku70/80 levels, and Ku-DNA-binding activity of the cells treated with or without antioxidant N-acetyl cysteine and caspase-3 inhibitor z-DEVED-fmk. As a result, β-carotene induced apoptosis (decrease in cell viability, increases in DNA fragmentation and apoptotic indices) and caspase-3 activation, but decreased Ku70/80 levels and Ku-DNA-binding activity. β-Carotene-induced alterations (increase in caspase-3 activity, decrease in Ku proteins) and apoptosis were inhibited by N-acetyl cysteine and z-DEVED-fmk. Increment of intracellular and mitochondrial ROS levels and loss of mitochondrial membrane potential were suppressed by N-acetyl cysteine, but not by z-DEVED-fmk in β-carotene-treated cells. Therefore, β-carotene-induced increases in ROS and caspase-3 activity may lead to reduction of Ku70/80 levels, which results in apoptosis in gastric cancer cells. Loss of Ku proteins might be the underlying mechanism for β-carotene-induced apoptosis in gastric cancer cells.     

A Ku80 fragment with dominant negative activity imparts a radiosensitive phenotype to CHO-K1 cells

DNA non-homologous end joining, the major mechanism for the repair of DNA double-strands breaks (DSB) in mammalian cells requires the DNA-dependent protein kinase (DNA-PK), a complex composed of a large catalytic subunit of 460 kDa (DNA-PKcs) and the heterodimer Ku70–Ku80 that binds to double-stranded DNA ends. Mutations in any of the three subunits of DNA-PK lead to extreme radiosensitivity and DSB repair deficiency. Here we show that the 283 C-terminal amino acids of Ku80 introduced into the Chinese hamster ovary cell line CHO-K1 have a dominant negative effect. Expression of Ku(449–732) in CHO cells was verified by northern blot analysis and resulted in decreased Ku-dependent DNA end-binding activity, a diminished capacity to repair DSBs as determined by pulsed field gel electrophoresis and decreased radioresistance determined by clonogenic survival. The stable modifications observed at the molecular and cellular level suggest that this fragment of Ku80 confers a dominant negative effect providing an important mechanism to sensitise radioresistant cells.     

Structural basis of importin-α-mediated nuclear transport for Ku70 and Ku80

Ku70 and Ku80 form a heterodimeric complex involved in multiple nuclear processes. This complex plays a key role in DNA repair due to its ability to bind DNA double-strand breaks and facilitate repair by the nonhomologous end-joining pathway. Ku70 and Ku80 have been proposed to contain bipartite and monopartite nuclear localization sequences (NLSs), respectively, that allow them to be translocated to the nucleus independently of each other via the classical importin-α (Impα)/importin-β-mediated nuclear import pathway. To determine the structural basis of the recognition of Ku70 and Ku80 proteins by Impα, we solved the crystal structures of the complexes of Impα with the peptides corresponding to the Ku70 and Ku80 NLSs. Our structural studies confirm the binding of the Ku80 NLS as a classical monopartite NLS but reveal an unexpected binding mode for Ku70 NLS with only one basic cluster bound to the receptor. Both Ku70 and Ku80 therefore contain monopartite NLSs, and sequences outside the basic cluster make favorable interactions with Impα, suggesting that this may be a general feature in monopartite NLSs. We show that the Ku70 NLS has a higher affinity for Impα than the Ku80 NLS, consistent with more extensive interactions in its N-terminal region. The prospect of nuclear import of Ku70 and Ku80 independently of each other provides a powerful regulatory mechanism for the function of the Ku70/Ku80 heterodimer and independent functions of the two proteins.     

Mutations in two Ku homologs define a DNA end-joining repair pathway in Saccharomyces cerevisiae.

DNA double-strand break (DSB) repair in mammalian cells is dependent on the Ku DNA binding protein complex. However, the mechanism of Ku-mediated repair is not understood. We discovered a Saccharomyces cerevisiae gene (KU80) that is structurally similar to the 80-kDa mammalian Ku subunit. Ku8O associates with the product of the HDF1 gene, forming the major DNA end-binding complex of yeast cells. DNA end binding was absent in ku80delta, hdf1delta, or ku80delta hdf1delta strains. Antisera specific for epitope tags on Ku80 and Hdf1 were used in supershift and immunodepletion experiments to show that both proteins are directly involved in DNA end binding. In vivo, the efficiency of two DNA end-joining processes were reduced >10-fold in ku8Odelta, hdfldelta, or ku80delta hdf1delta strains: repair of linear plasmid DNA and repair of an HO endonuclease-induced chromosomal DSB. These DNA-joining defects correlated with DNA damage sensitivity, because ku80delta and hdf1delta strains were also sensitive to methylmethane sulfonate (MMS). Ku-dependent repair is distinct from homologous recombination, because deletion of KU80 and HDF1 increased the MMS sensitivity of rad52delta. Interestingly, rad5Odelta, also shown here to be defective in end joining, was epistatic with Ku mutations for MMS repair and end joining. Therefore, Ku and Rad50 participate in an end-joining pathway that is distinct from homologous recombinational repair. Yeast DNA end joining is functionally analogous to DSB repair and V(D)J recombination in mammalian cells.     

Accumulation of Ku70 at DNA double-strand breaks in living epithelial cells

Ku70 and Ku80 play an essential role in the DNA double-strand break (DSB) repair pathway, i.e., nonhomologous DNA-end-joining (NHEJ). No accumulation mechanisms of Ku70 at DSBs have been clarified in detail, although the accumulation mechanism of Ku70 at DSBs plays key roles in regulating the NHEJ activity. Here, we show the essential domains for the accumulation and function of Ku70 at DSBs in living lung epithelial cells. Our results showed that EGFP-Ku70 accumulation at DSBs began immediately after irradiation. Our findings demonstrate that three domains of Ku70, i.e., the α/β, DNA-binding, and Ku80-binding domains, but not the SAP domain, are necessary for the accumulation at or recognition of DSBs in the early stage after irradiation. Moreover, our findings demonstrate that the leucine at amino acid 385 of Ku70 in the Ku80-binding domain, but not the three target amino acids for acetylation in the DNA-binding domain, is involved in the localization and accumulation of Ku70 at DSBs. Furthermore, accumulations of XRCC4 and XLF, but not that of Artemis, at DSBs are dependent on the presence of Ku70. These findings suggest that Artemis can work in not only the Ku-dependent repair process, but also the Ku-independent process at DSBs in living epithelial cells.     

An SCF complex containing Fbxl12 mediates DNA damage-induced Ku80 ubiquitylation

The Ku heterodimer, composed of Ku70 and Ku80, is the initiating factor of the nonhomologous end joining (NHEJ) double-strand break (DSB) repair pathway. Ku is also thought to impede the homologous recombination (HR) repair pathway via inhibition of DNA end resection. Using the cell-free Xenopus laevis egg extract system, we had previously discovered that Ku80 becomes polyubiquitylated upon binding to DSBs, leading to its removal from DNA and subsequent proteasomal degradation. Here we show that the Skp1-Cul1-F box (SCF) E3 ubiquitin ligase complex is required for Ku80 ubiquitylation and removal from DNA. A screen for DSB-binding F box proteins revealed that the F box protein Fbxl12 was recruited to DNA in a DSB- and Ku-sensitive manner. Immunodepletion of Fbxl12 prevented Cul1 and Skp1 binding to DSBs and Ku80 ubiquitylation, indicating that Fbxl12 is the F box protein responsible for Ku80 substrate recognition. Unlike typical F box proteins, the F box of Fbxl12 was essential for binding to both Skp1 and its substrate Ku80. Besides Fbxl12, six other chromatin-binding F box proteins were identified in our screen of a subset of Xenopus F box proteins: β-TrCP, Fbh1, Fbxl19, Fbxo24, Fbxo28 and Kdm2b. Our study unveils a novel function for the SCF ubiquitin ligase in regulating the dynamic interaction between DNA repair machineries and DSBs.