Persistently bound Ku at DNA ends attenuates DNA end resection and homologous recombination

DNA double strand breaks (DSBs) are repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). The DNA cell cycle stage and resection of the DSB ends are two key mechanisms which are believed to push DSB repair to the HR pathway. Here, we show that the NHEJ factor Ku80 associates with DSBs in S phase, when HR is thought to be the preferred repair pathway, and its dynamics/kinetics at DSBs is similar to those observed for Ku80 in non-S phase in mammalian cells. A Ku homolog from Mycobacterium tuberculosis binds to and is retained at DSBs in S phase and was used as a tool to determine if blocking DNA ends affects end resection and HR in mammalian cells. A decrease in DNA end resection, as marked by IR-induced RPA, BrdU, and Rad51 focus formation, and HR are observed when Ku deficient rodent cells are complemented with Mt-Ku. Together, this data suggests that Ku70/80 binds to DSBs in all cell cycle stages and is likely actively displaced from DSB ends to free the DNA ends for DNA end resection and thus HR to occur.     

KARP-1 works as a heterodimer with Ku70, but the function of KARP-1 cannot perfectly replace that of Ku80 in DSB repair

Ku, the heterodimer of Ku70 and Ku80, plays an essential role in the DNA double-strand break (DSB) repair pathway, i.e., non-homologous end-joining (NHEJ). Two isoforms of Ku80 encoded by the same genes, namely, Ku80 and KARP-1 are expressed and function in primate cells, but not in rodent cells. Ku80 works as a heterodimer with Ku70. However, it is not yet clear whether KARP-1 forms a heterodimer with Ku70 and works as a heterodimer. Although KARP-1 appears to work in NHEJ, its physiological role remains unclear. In this study, we established and characterized EGFP-KARP-1-expressing xrs-6 cell lines, EGFP-KARP-1/xrs-6. We found that nuclear localization signal (NLS) of KARP-1 is localized in the C-terminal region. Our data showed that KARP-1 localizes within the nucleus in NLS-dependent and NLS-independent manner and forms a heterodimer with Ku70, and stabilizes Ku70. On the other hand, EGFP-KARP-1 could not perfectly complement the radiosensitivity and DSB repair activity of Ku80-deficient xrs-6 cells. Furthermore, KARP-1 could not accumulate at DSBs faster than Ku80, although EGFP-KARP-1 accumulates at DSBs. Our data demonstrate that the function of KARP-1 could not perfectly replace that of Ku80 in DSB repair, although KARP-1 has some biochemical properties, which resemble those of Ku80, and works as a heterodimer with Ku70. On the other hand, the number of EGFP-KARP-1-expressing xrs-6 cells showing pan-nuclear γ-H2AX staining significantly increases following X-irradiation, suggesting that KARP-1 may have a novel role in DSB response.     

Phosphorylation of Ku dictates DNA double-strand break (DSB) repair pathway choice in S phase

Multiple DNA double-strand break (DSB) repair pathways are active in S phase of the cell cycle; however, DSBs are primarily repaired by homologous recombination (HR) in this cell cycle phase. As the non-homologous end-joining (NHEJ) factor, Ku70/80 (Ku), is quickly recruited to DSBs in S phase, we hypothesized that an orchestrated mechanism modulates pathway choice between HR and NHEJ via displacement of the Ku heterodimer from DSBs to allow HR. Here, we provide evidence that phosphorylation at a cluster of sites in the junction of the pillar and bridge regions of Ku70 mediates the dissociation of Ku from DSBs. Mimicking phosphorylation at these sites reduces Ku''s affinity for DSB ends, suggesting that phosphorylation of Ku70 induces a conformational change responsible for the dissociation of the Ku heterodimer from DNA ends. Ablating phosphorylation of Ku70 leads to the sustained retention of Ku at DSBs, resulting in a significant decrease in DNA end resection and HR, specifically in S phase. This decrease in HR is specific as these phosphorylation sites are not required for NHEJ. Our results demonstrate that the phosphorylation-mediated dissociation of Ku70/80 from DSBs frees DNA ends, allowing the initiation of HR in S phase and providing a mechanism of DSB repair pathway choice in mammalian cells.     

The dynamics of Ku70/80 and DNA-PKcs at DSBs induced by ionizing radiation is dependent on the complexity of damage

DNA double-strand breaks (DSBs) are biologically one of the most important cellular lesions and possess varying degrees of chemical complexity. The notion that the repairability of more chemically complex DSBs is inefficient led to the concept that the extent of DSB complexity underlies the severity of the biological consequences. The repair of DSBs by non-homologous end joining (NHEJ) has been extensively studied but it remains unknown whether more complex DSBs require a different sub-set of NHEJ protein for their repair compared with simple DSBs. To address this, we have induced DSBs in fluorescently tagged mammalian cells (Ku80-EGFP, DNA-PKcs-YFP or XRCC4-GFP, key proteins in NHEJ) using ultra-soft X-rays (USX) or multi-photon near infrared (NIR) laser irradiation. We have shown in real-time that simple DSBs, induced by USX or NIR microbeam irradiation, are repaired rapidly involving Ku70/80 and XRCC4/Ligase IV/XLF. In contrast, DSBs with greater chemical complexity are repaired slowly involving not only Ku70/80 and XRCC4/Ligase IV/XLF but also DNA-PKcs. Ataxia telangiectasia-mutated inhibition only retards repair of the more chemically complex DSBs which require DNA-PKcs. In summary, the repair of DSBs by NHEJ is highly regulated with pathway choice and kinetics of repair dependent on the chemical complexity of the DSB.     

Ablating putative Ku70 phosphorylation sites results in defective DNA damage repair and spontaneous induction of hepatocellular carcinoma

Multiple pathways mediate the repair of DNA double-strand breaks (DSBs), with numerous mechanisms responsible for driving choice between the pathways. Previously, we reported that mutating five putative phosphorylation sites on the non-homologous end joining (NHEJ) factor, Ku70, results in sustained retention of human Ku70/80 at DSB ends and attenuation of DSB repair via homologous recombination (HR). In this study, we generated a knock-in mouse, in which the three conserved putative phosphorylation sites of Ku70 were mutated to alanine to ablate potential phosphorylation (Ku70^3A/3A), in order to examine if disrupting DSB repair pathway choice by modulating Ku70/80 dynamics at DSB ends results in enhanced genomic instability and tumorigenesis. The Ku70^3A/3A mice developed spontaneous and have accelerated chemical-induced hepatocellular carcinoma (HCC) compared to wild-type (Ku70^+/+) littermates. The HCC tumors from the Ku70^3A/3A mice have increased γH2AX and 8-oxo-G staining, suggesting decreased DNA repair. Spontaneous transformed cell lines from Ku70^3A/3A mice are more radiosensitive, have a significant decrease in DNA end resection, and are more sensitive to the DNA cross-linking agent mitomycin C compared to cells from Ku70^+/+ littermates. Collectively, these findings demonstrate that mutating the putative Ku70 phosphorylation sites results in defective DNA damage repair and disruption of this process drives genomic instability and accelerated development of HCC.     

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.     

The anaphase promoting complex promotes NHEJ repair through stabilizing Ku80 at DNA damage sites

Double-strand breaks (DSBs) are repaired through two major pathways, homology-directed recombination (HDR) and non-homologous end joining (NHEJ). The choice between these two pathways is largely influenced by cell cycle phases. HDR can occur only in S/G2 when sister chromatid can provide homologous templates, whereas NHEJ can take place in all phases of the cell cycle except mitosis. Central to NHEJ repair is the Ku70/80 heterodimer which forms a ring structure that binds DSB ends and serves as a platform to recruit factors involved in NHEJ. Upon completion of NHEJ repair, DNA double strand-encircling Ku dimers have to be removed. The removal depends on ubiquitylation and proteasomal degradation of Ku80 by the ubiquitin E3 ligases RNF8. Here we report that RNF8 is a substrate of APC^Cdh1 and the latter keeps RNF8 level in check at DSBs to prevent premature turnover of Ku80.     

Expression and chromosome location of hamster Ku70 and Ku80

Ku proteins play an important role in DNA double-strand break (DSB) repair, chromosome maintenance, and growth regulation. To understand the fundamental characteristics of Ku proteins, we examined the electrophoretic mobility and expression of hamster Ku70 and Ku80 and determined the chromosome locations of their genes. The electrophoretic mobility of hamster Ku proteins are different from that of human Ku proteins. No significant changes in the quantity of Ku proteins were observed in CHO-K1 cells treated with 10 Gy of ionizing radiation, suggesting that both proteins are expressed constitutively in amounts adequate to repair DNA DSBs. The chromosome locations of the Ku genes were determined by direct R-banding fluorescence in situ hybridization. The Ku70 gene was localized to Syrian hamster chromosome 4qa4.1--> qa4.2 and Chinese hamster chromosome 2p3.1, and the Ku80 gene was localized to Syrian hamster chromosome 4qb5--> qb6.1 and Chinese hamster chromosome 2p3.5-->p3.6. These results provide clues to the biological functions of Ku, as well as useful information for constructing comparative chromosome maps between hamsters and other mammalian species, including human, mouse, and rat.     

Saccharomyces cerevisiae Ku70 potentiates illegitimate DNA double-strand break repair and serves as a barrier to error-prone DNA repair pathways.

Ku, a heterodimer of polypeptides of approximately 70 kDa and 80 kDa (Ku70 and Ku80, respectively), binds avidly to DNA double-strand breaks (DSBs). Mammalian cells defective in Ku are hypersensitive to ionizing radiation due to a deficiency in DSB repair. Here, we show that the simple inactivation of the Saccharomyces cerevisiae Ku70 homologue (Yku70p), does not lead to increased radiosensitivity. However, yku70 mutations enhance the radiosensitivity of rad52 strains, which are deficient in homologous recombination. Through establishing a rapid and reproducible in vivo plasmid rejoining assay, we show that Yku70p plays a crucial role in the repair of DSBs bearing cohesive termini. Whereas this damage is repaired accurately in YKU70 backgrounds, in yku70 mutant strains terminal deletions of up to several hundred bp occur before ligation ensues. Interestingly, this error-prone DNA repair pathway utilizes short homologies between the two recombining molecules and is thus highly reminiscent of a predominant form of DSB repair that operates in vertebrates. These data therefore provide evidence for two distinct and evolutionarily conserved illegitimate recombination pathways. One of these is accurate and Yku70p-dependent, whereas the other is error-prone and Yku70-independent. Furthermore, our studies suggest that Yku70 promotes genomic stability both by promoting accurate DNA repair and by serving as a barrier to error-prone repair processes.     

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.     

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.     

Rapid recruitment of BRCA1 to DNA double-strand breaks is dependent on its association with Ku80

BRCA1 is the first susceptibility gene to be linked to breast and ovarian cancers. Although mounting evidence has indicated that BRCA1 participates in DNA double-strand break (DSB) repair pathways, its precise mechanism is still unclear. Here, we analyzed the in situ response of BRCA1 at DSBs produced by laser microirradiation. The amino (N)- and carboxyl (C)-terminal fragments of BRCA1 accumulated independently at DSBs with distinct kinetics. The N-terminal BRCA1 fragment accumulated immediately after laser irradiation at DSBs and dissociated rapidly. In contrast, the C-terminal fragment of BRCA1 accumulated more slowly at DSBs but remained at the sites. Interestingly, rapid accumulation of the BRCA1 N terminus, but not the C terminus, at DSBs depended on Ku80, which functions in the nonhomologous end-joining (NHEJ) pathway, independently of BARD1, which binds to the N terminus of BRCA1. Two small regions in the N terminus of BRCA1 independently accumulated at DSBs and interacted with Ku80. Missense mutations found within the N terminus of BRCA1 in cancers significantly changed the kinetics of its accumulation at DSBs. A P142H mutant failed to associate with Ku80 and restore resistance to irradiation in BRCA1-deficient cells. These might provide a molecular basis of the involvement of BRCA1 in the NHEJ pathway of the DSB repair process.     

A novel small molecule inhibitor of the DNA repair protein Ku70/80

Non-Homologous End-Joining (NHEJ) is the predominant pathway for the repair of DNA double strand breaks (DSBs) in human cells. The NHEJ pathway is frequently upregulated in several solid cancers as a compensatory mechanism for a separate DSB repair defect or for innate genomic instability, making this pathway a powerful target for synthetic lethality approaches. In addition, NHEJ reduces the efficacy of cancer treatment modalities which rely on the introduction of DSBs, like radiation therapy or genotoxic chemotherapy. Consequently, inhibition of the NHEJ pathway can modulate a radiation- or chemo-refractory disease presentation. The Ku70/80 heterodimer protein plays a pivotal role in the NHEJ process. It possesses a ring-shaped structure with high affinity for DSBs and serves as the first responder and central scaffold around which the rest of the repair complex is assembled. Because of this central position, the Ku70/80 dimer is a logical target for the disruption of the entire NHEJ pathway. Surprisingly, specific inhibitors of the Ku70/80 heterodimer are currently not available. We here describe an in silico, pocket-based drug discovery methodology utilizing the crystal structure of the Ku70/80 heterodimer. We identified a novel putative small molecule binding pocket and selected several potential inhibitors by computational screening. Subsequent biological screening resulted in the first identification of a compound with confirmed Ku-inhibitory activity in the low micro-molar range, capable of disrupting the binding of Ku70/80 to DNA substrates and impairing Ku-dependent activation of another NHEJ factor, the DNA-PK_CS kinase. Importantly, this compound synergistically sensitized human cell lines to radiation treatment, indicating a clear potential to diminish DSB repair. The chemical scaffold we here describe can be utilized as a lead-generating platform for the design and development of a novel class of anti-cancer agents.     

Increased repair of gamma-induced DNA double-strand breaks at lower dose-rate in CHO cells

DNA double-strand breaks (DSBs) are highly cell damaging. We asked whether for a given dose a longer irradiation time would be advantageous for the repair of DSBs. Varying the gamma-irradiation dose and its delivery time (0.05 Gy/min low dose-rate (LDR) compared with 3.5 Gy/min high dose-rate), confluent Chinese hamster ovary cells (CHO-K1) and Ku80 mutant cells (xrs-6) deficient in nonhomologous end-joining (NHEJ) were irradiated in agarose plugs at room temperature using a cesium-137 gamma-ray source. We used pulsed-field gel electrophoresis (PFGE) to measure DSBs in terms of the fraction of activity released (FAR). At LDR, one third of DSBs were repaired in CHO-K1 but not in xrs-6 cells, indicating the involvement of NHEJ in the repair of gamma-induced DSBs at a prolonged irradiation incubation time. To improve DSB measurements, we introduced in our PFGE protocol an antioxidant at the cell lysis step, thus avoiding free-radical side reactions on DNA and spurious DSBs. Addition of the metal chelator deferoxamine (DFO) decreased more efficiently the basal DSB level than did reduced glutathione (GSH), showing that measuring DSBs in the absence of DFO reduces precision and underestimates the role of NHEJ in the dose-rate effect on DSB yield.     

Heterozygous inactivation of human Ku70/Ku86 heterodimer does not affect cell growth, double-strand break repair, or genome integrity

Ku, the heterodimer of Ku70 and Ku86, plays crucial roles in non-homologous end-joining (NHEJ), a major pathway for repairing DNA double-strand breaks (DSBs) in mammalian cells. It has recently been reported that heterozygous disruption of the human KU86 locus results in haploinsufficient phenotypes, including retarded growth, increased radiosensitivity, elevated p53 levels and shortened telomeres. In this paper, however, we show that heterozygous inactivation of either the KU70 or KU86 gene does not cause any defects in cell proliferation or DSB repair in human somatic cells. Moreover, although these heterozygous cell lines express reduced levels of both Ku70 and Ku86, they appear to maintain overall genome integrity with no elevated p53 levels or telomere shortening. These results clearly indicate that Ku haploinsufficiency is not a commonly observed phenomenon in human cells. Our data also suggest that the impact of KU70/KU86 mutations on telomere metabolism varies between cell types in humans.     

Ku80 removal from DNA through double strand break-induced ubiquitylation

The Ku70/Ku80 heterodimer, or Ku, is the central component of the nonhomologous end joining (NHEJ) pathway of double strand break (DSB) repair. Because Ku forms a ring through which the DSB threads, it likely becomes topologically attached to DNA during repair. The mechanism for its removal was unknown. Using a method to identify proteins recruited to DSBs in Xenopus laevis egg extract, we show that DSB-containing DNAs accumulate members of the Skp1-Cul1-F-box complex and K48-linked polyubiquitylated proteins in addition to known repair proteins. We demonstrate that Ku80 is degraded in response to DSBs in a ubiquitin-mediated manner. Strikingly, K48-linked polyubiquitylation, but not proteasomal degradation, is required for the efficient removal of Ku80 from DNA. This removal is DNA length dependent, as Ku80 is retained on duplex oligonucleotides. Finally, NHEJ completion and removal of Ku80 from DNA are independent from one another. We propose that DSB-induced ubiquitylation of Ku80 provides a mechanism to efficiently eliminate Ku from DNA for pre- and postrepair processes.     

Accumulation of Ku80 proteins at DNA double-strand breaks in living cells

Ku plays a key role in multiple nuclear processes, e.g., DNA double-strand break (DSB) repair. The regulation mechanism of the localizations of Ku70 and Ku80 plays a key role in regulating the multiple functions of Ku. Although numerous biochemical studies in vitro have elucidated the DNA binding mechanism of Ku, no accumulation mechanisms of Ku70 and Ku80 at DSBs have been clarified in detail in vivo. In this study, we examined the accumulation mechanism of Ku80 at DSBs in living cells. EGFP-Ku80 accumulation at DSBs began immediately after irradiation. On the other hand, our data show that Ku70 alone, which has DNA binding activity independent of Ku80, cannot accumulate at the DSBs, whereas Ku70 bound to Ku80 can. The deletion of the C-terminal DNA-PKcs-binding domain and the mutation at the SUMOylation site of Ku80 had no effect on Ku80 accumulation. Unexpectedly, N-terminal deletion mutants of Ku80 fully lost their accumulation activity, although the mutants retained their Ku70 binding activity. Altogether, these data demonstrate that Ku80 is essential for Ku70 accumulation at DSBs. Furthermore, three domains of Ku80, i.e., the N-terminal α/β, the DNA-binding, and Ku70-binding domains, seem to necessary for the accumulation at or recognition of DSBs in the early stage after irradiation.