Ku Subunits Research Articles

Structural and functional basis of inositol hexaphosphate stimulation of NHEJ through stabilization of Ku-XLF interaction.

The classical Non-Homologous End Joining (c-NHEJ) pathway is the predominant process in mammals for repairing endogenous, accidental or programmed DNA Double-Strand Breaks. c-NHEJ is regulated by several accessory factors, post-translational modifications, endogenous chemical agents and metabolites. The metabolite inositol-hexaphosphate (IP6) stimulates c-NHEJ by interacting with the Ku70-Ku80 heterodimer (Ku). We report cryo-EM structures of apo- and DNA-bound Ku in complex with IP6, at 3.5 Å and 2.74 Å resolutions respectively, and an X-ray crystallography structure of a Ku in complex with DNA and IP6 at 3.7 Å. The Ku-IP6 interaction is mediated predominantly via salt bridges at the interface of the Ku70 and Ku80 subunits. This interaction is distant from the DNA, DNA-PKcs, APLF and PAXX binding sites and in close proximity to XLF binding site. Biophysical experiments show that IP6 binding increases the thermal stability of Ku by 2°C in a DNA-dependent manner, stabilizes Ku on DNA and enhances XLF affinity for Ku. In cells, selected mutagenesis of the IP6 binding pocket reduces both Ku accrual at damaged sites and XLF enrolment in the NHEJ complex, which translate into a lower end-joining efficiency. Thus, this study defines the molecular bases of the IP6 metabolite stimulatory effect on the c-NHEJ repair activity.

Acetylation of the nuclear localization signal in Ku70 diminishes the interaction with importin-α

Proteins are functionally regulated by various types of posttranslational modifications (PTMs). Ku, a heterodimer complex of Ku70 and Ku80 subunits, participates in DNA repair processes. Ku is distributed not only in the nucleus but also in the cytoplasm, suggesting that the function of Ku is regulated by its subcellular localization. Although Ku70 undergoes PTMs including phosphorylation or acetylation, it remains unknown whether the PTMs of Ku70 affect the subcellular localization of Ku. Using a cell-free pull-down assay technique, we show that Nε-acetylation of lysine residues in the synthetic peptide matched to Ku70's nuclear localization signal (NLS) reduces the peptide's interaction with the nuclear transport factor importin-α. The reduced interaction by acetylation was supported by molecular simulation analysis. In addition, when expressed in the endogenous Ku80-defective Chinese hamster ovary xrs-6 cells, some full-size human Ku70 mutants with substitutions of glutamine, a possible structural mimetic of Nε-acetyl–lysine, for lysine at the specific NLS positions exhibited no nuclear distribution. These findings imply that acetylation of particular lysine residues in the Ku70 NLS regulates nuclear localization of Ku.

Transcriptome analysis of HEK 293T cells revealed different significance of the depletion of DNA-dependent protein kinase subunits, Ku70, Ku80, and DNA-PKcs

DNA-dependent protein kinase (DNA-PK) is a key player in the NHEJ repair pathway. DNA-PK and its subunits, Ku70, Ku80, and catalytic subunit (DNA-PKcs), also participate in other cellular processes; however, there are still no systemic data on the effect of depletion of Ku70, Ku80 and DNA-PKcs on cell functions in the same cell line. Here, we analyzed transcriptome changes in HEK 293T cells after depletion of each DNA-PK subunit. Depletion of various DNA-PK subunits resulted in dramatic differences in the number of differentially expressed genes: only 7 genes changed more than 2-fold in DNA-PKcs-deficient cells, 29 genes in Ku80-deficient, 219 genes in Ku70-deficient. All DNA-PKcs-dependent genes were stress-related and depended on both Ku70 and Ku80. Two-thirds of Ku80-dependent genes were also differentially expressed in the Ku70-deficient line. Most Ku70-dependent genes were altered exclusively in Ku70-depleted cells, indicating that Ku70 is involved in the regulation of more processes than Ku80. GO enrichment analysis showed the effect of Ku70 knockdown on cell adhesion and matrix organization, protein degradation, cell proliferation, and differentiation. Depletion of Ku70, but not Ku80, provided greater cell motility and disassembly of cell-cell contacts. These data clearly indicate that Ku70 is more functionally important for the cell life than DNA-PKcs and even Ku80.

Genetic interaction between the non-homologous end-joining factors during B and T lymphocyte development: In vivo mouse models.

Non-homologous end joining (NHEJ) is the main DNA repair mechanism for the repair of double-strand breaks (DSBs) throughout the course of the cell cycle. DSBs are generated in developing B and T lymphocytes during V(D)J recombination to increase the repertoire of B and T cell receptors. DSBs are also generated during the class switch recombination (CSR) process in mature B lymphocytes, providing distinct effector functions of antibody heavy chain constant regions. Thus, NHEJ is important for both V(D)J recombination and CSR. NHEJ comprises core Ku70 and Ku80 subunits that form the Ku heterodimer, which binds DSBs and promotes the recruitment of accessory factors (e.g., DNA-PKcs, Artemis, PAXX, MRI) and downstream core factors (XLF, Lig4 and XRCC4). In recent decades, new NHEJ proteins have been reported, increasing complexity of this molecular pathway. Numerous in vivo mouse models have been generated and characterized to identify the interplay of NHEJ factors and their role in development of adaptive immune system. This review summarizes the currently available mouse models lacking one or several NHEJ factors, with a particular focus on early B cell development. We also underline genetic interactions and redundancy in the NHEJ pathway in mice.

Analysis of RNA binding properties of human Ku protein reveals its interactions with 7SK snRNA and protein components of 7SK snRNP complex

Human Ku heterodimeric protein composed of Ku70 and Ku80 subunits plays an important role in the non-homologous end-joining DNA repair pathway as a sensor of double strand DNA breaks. Ku is also involved in numerous cellular processes, and in some of them it acts in an RNA-dependent manner. However, RNA binding properties of the human Ku have not been well studied. Here we have analyzed interactions of a recombinant Ku heterodimer with a set of RNAs of various structure as well as eCLIP (enhanced crosslinking and immunoprecipitation) data for human Ku70. As a result, we have proposed a consensus RNA structure preferable for the Ku binding that is a hairpin possessing a bulge just near GpG sequence-containing terminal loop. 7SK snRNA is a scaffold for a ribonucleoprotein complex (7SK snRNP), which is known to participate in transcription regulation. We have shown that the recombinant Ku specifically binds a G-rich loop of hairpin 1 within 7SK snRNA. Moreover, Ku protein has been co-precipitated from HEK 293T cells with endogenous 7SK snRNA and such proteins included in 7SK snRNP as HEXIM1, Cdk9 and CTIP2. Ku and Cdk9 binding is found to be RNA-independent, meanwhile HEXIM1 and Ku co-precipitation depended on the presence of intact 7SK snRNA. The latter result has been confirmed using recombinant HEXIM1 and Ku proteins. Colocalization of Ku and CTIP2 was additionally confirmed by confocal microscopy. These results allow us to propose human Ku as a new component of the 7SK snRNP complex.

Chronic fetal hypoxia disrupts the peri-conceptual environment in next-generation adult female rats.

Key points Exposure to chronic hypoxia during gestation influences long‐term health and development, including reproductive capacity, across generations.If the peri‐conceptual environment in the developing oviduct is affected by gestational hypoxia, then this could have implications for later fertility and the health of future generations.In the present study, we show that the oviducts of female rats exposed to chronic hypoxia in utero have reduced telomere length, decreased mitochondrial DNA biogenesis and increased oxidative stressThe results of the present study show that exposure to chronic gestational hypoxia leads to accelerated ageing of the oviduct in early adulthood and they help us understand how exposure to hypoxia during development could influence reproductive health across generations. Exposure to chronic hypoxia during fetal development has important effects on immediate and long‐term outcomes in offspring. Adverse impacts in adult offspring include impairment of cardiovascular function, metabolic derangement and accelerated ovarian ageing. However, it is not known whether other aspects of the female reproductive system may be similarly affected. In the present study, we examined the impact of chronic gestational hypoxia on the developing oviduct. Wistar rat dams were randomized to either normoxia (21%) or hypoxia (13%) from day 6 post‐mating until delivery. Post‐delivery female offspring were maintained in normoxia until 4 months of age. Oviductal gene expression was assayed at the RNA (quantitative RT‐PCR) and protein (western blotting) levels. Oviductal telomere length was assayed using Southern blotting. Oviductal telomere length was reduced in the gestational hypoxia‐exposed animals compared to normoxic controls (P < 0.01). This was associated with a specific post‐transcriptional reduction in the KU70 subunit of DNA‐pk in the gestational hypoxia‐exposed group (P < 0.05). Gestational hypoxia‐exposed oviducts also showed evidence of decreased mitochondrial DNA biogenesis, reduced mtDNA copy number (P < 0.05) and reduced gene expression of Tfam (P < 0.05) and Pgc1α (P < 0.05). In the hypoxia‐exposed oviducts, there was upregulation of mitochondrial‐specific anti‐oxidant defence enzymes (MnSOD; P < 0.01). Exposure to chronic gestational hypoxia leads to accelerated ageing of the oviduct in adulthood. The oviduct plays a central role in early development as the site of gamete transport, syngamy, and early development; hence, accelerated ageing of the oviductal environment could have important implications for fertility and the health of future generations.

Synthetic lethality between DNA repair factors Xlf and Paxx is rescued by inactivation of Trp53

Non-homologous end joining (NHEJ) is a DNA repair pathway that senses, processes and ligates DNA double-strand breaks (DSBs) throughout the cell cycle. During NHEJ, core Ku70 and Ku80 subunits bind DSBs as a heterodimer and promote further recruitment of accessory factors (e.g., PAXX, Mri, DNA-PKcs, Artemis) and downstream core subunits XRCC4 and DNA ligase 4 (Lig4). Inactivation of Ku70 or Ku80 genes in mice results in immunodeficiency and high levels of genomic instability; deletion of individual Dna-pkcs, Xlf, Paxx or Mri genes results in viable mice with no or modest DNA repair defects. However, combined inactivation of either Xlf and Dna-pkcs, or Xlf and Paxx, or Xlf and Mri, leads to synthetic lethality in mice, which correlates with increased levels of apoptosis in the central nervous system. Here, we demonstrated that inactivation of pro-apoptotic factor Trp53 rescues embryonic lethality of Xlf−/−Paxx−/− and Xlf−/−Dna-pkcs−/− double knockout mice. Moreover, combined inactivation of Paxx and Dna-pkcs results in live-born fertile Paxx−/−Dna-pkcs−/− mice indistinguishable from Dna-pkcs−/− knockout controls.

Ets-1 interacts through a similar binding interface with Ku70 and Poly (ADP-Ribose) Polymerase-1.

The Ets-1 transcription factor plays an important role in various physiological and pathological processes. These diverse roles of Ets-1 are likely to depend on its interaction proteins. We have previously showed that Ets-1 interacted with DNA-dependent protein kinase (DNA-PK) complex including its regulatory subunits, Ku70 and Ku86 and with poly (ADP-ribose) polymerase-1 (PARP-1). In this study, the binding domains for the interaction between Ets-1 and these proteins were reported. We demonstrated that the interaction of Ets-1 with DNA-PK was mediated through the Ku70 subunit and was mapped to the C-terminal region of Ets-1 and the C-terminal part of Ku70 including SAP domain. The interactive domains between Ets-1 and PARP-1 have been mapped to the C-terminal region of Ets-1 and the BRCA1 carboxy-terminal (BRCT) domain of PARP-1. The results presented in this study may advance our understanding of the functional link between Ets-1 and its interaction partners, DNA-PK and PARP-1.

Characterization of HIV-1 integrase interaction with human Ku70 protein and initial implications for drug targeting

Human Ku70/Ku80 protein is known to influence HIV-1 replication. One of the possible reasons may be the protection of integrase from proteasomal degradation by Ku70 subunit. We demonstrated that recombinant HIV-1 integrase and Ku70 form a stable complex, while no interaction of Ku70 with integrase from prototype foamy virus was observed. By analyzing protein subdomains we determined two binding sites in the structure of both Ku70 and integrase: the 51–160 a.a. region of integrase interacts with residues 251–438 of Ku70, whereas Ku70 N-terminal domain (1–250 a.a.) contacts an α6-helix in the 200–220 a.a. integrase region. Single substitutions within integrase (E212A or L213A) block the interaction with Ku70 thus indicating that the binding site formed by the 200–220 a.a. integrase region is crucial for complex formation. E212A/L213A substitutions decreased the integrase capacity to bind Ku70 in HEK293T cells. A conjugate of 2′-ОMe-GGUUUUUGUGU oligonucleotide with eosin is shown by molecular modeling to shield integrase residues E212/L213 and is effective in blocking complex formation of Ku70 with integrase what makes the complex between α6-helix and Ku70(1–250) a possible target for drug development.

Human Ku70 protein binds hairpin RNA and double stranded DNA through two different sites

Human protein Ku usually functions in the cell as a complex of two subunits, Ku70 and Ku80. The Ku heterodimer plays a key role in the non-homologous end joining DNA repair pathway by specifically recognizing the DNA ends at the site of the lesion. The binding of the Ku heterodimer to DNA has been well-studied, and its interactions with RNA have been also described. However, Ku70 subunit is known to have independent DNA binding capability, which is less characterized. RNA binding properties of Ku70 have not been yet specially studied. We have prepared recombinant full-length Ku70 and a set of its truncated mutants in E. coli, and studied their interactions with nucleic acids of various structures: linear single- and double-stranded DNA and RNA, as well as closed circular DNA and hairpin RNA. Ku70 has demonstrated a high affinity binding to double stranded DNA and hairpin RNA with a certain structure only. Interestingly, in contrast to the Ku heterodimer, Ku70 is found to interact with closed circular DNA. We also show for the first time that Ku70 employs two different sites for DNA and RNA binding. The double-stranded DNA is recognized by the C-terminal part of Ku70 including SAP domain as it has been earlier demonstrated, whereas hairpin RNA binding is provided by amino acids 251–438.

PICT-1 is a key nucleolar sensor in DNA damage response signaling that regulates apoptosis through the RPL11-MDM2-p53 pathway.

PICT-1 is an essential ribosome biogenesis factor whose loss induces p53 accumulation and apoptosis. Here, we show that DNA damage changes PICT-1 localization and decreases PICT-1 protein levels via the proteasome pathway. Two important phosphatidylinositol 3-kinase-like kinases (PIKKs), ataxia-telangiectasia mutated (ATM) and the Ku70 subunit of DNA-dependent protein kinase (DNA-PK), co-localize and interact with PICT-1 in the nucleolus. Computational prediction of phosphorylation sites and detection using an anti-phospho-substrate antibody suggest that PICT-1 might be a substrate of PIKKs. PICT-1 S233 and T289 were identified as the key phosphorylation sites in this pathway, as mutating both to alanine abolished UVB-induced increase of PICT-1 phosporylation. Inhibition of PIKKs or ATM (with wortmannin and KU55933, respectively) prevented the agglomeration and degradation of PICT-1, suggesting that ATM is a key regulator of PICT-1. PICT-1(S233A, T289A) demonstrated marked resistance to DNA damage-induced agglomeration and loss of PICT-1. Phosphomimetic PICT-1 (S233D, T289D) showed a different nuclear distribution and was more rapidly degraded after DNA damage than wild-type PICT-1. Furthermore, both phosphorylation and degradation of PICT-1 released RPL11 from the nucleolus to the nucleoplasm, increased binding of RPL11 to MDM2, and promoted p53 accumulation and apoptosis in an ATM-dependent manner after DNA damage. These data indicate that PICT-1 is a major nucleolar sensor of the DNA damage repair response and an important upstream regulator of p53 via the RPL11-MDM2-p53 pathway.

Phosphorylation of Ku70 subunit by cell cycle kinases modulates the replication related function of Ku heterodimer.

The Ku protein, a heterodimer of Ku70 and Ku80, binds to chromosomal replication origins maximally at G1-phase and plays an essential role in assembly of origin recognition complex. However, the mechanism regulating such a critical periodic activity of Ku remained unknown. Here, we establish human Ku70 as a novel target of cyclin B1-Cdk1, which phosphorylates it in a Cy-motif dependent manner. Interestingly, cyclin E1- and A2-Cdk2 also phosphorylate Ku70, and as a result, the protein remains in a phosphorylated state during S-M phases of cell cycle. Intriguingly, the phosphorylation of Ku70 by cyclin-Cdks abolishes the interaction of Ku protein with replication origin due to disruption of the dimer. Furthermore, Ku70 is dephosphorylated in G1-phase, when Ku interacts with replication origin maximally. Strikingly, the over-expression of Ku70 with non-phosphorylable Cdk targets enhances the episomal replication of Ors8 origin and induces rereplication in HeLa cells, substantiating a preventive role of Ku phosphorylation in premature and untimely licensing of replication origin. Therefore, periodic phosphorylation of Ku70 by cyclin-Cdks prevents the interaction of Ku with replication origin after initiation events in S-phase and the dephosphorylation at the end of mitosis facilitates its participation in pre-replication complex formation.

Ku antigen displays the AP lyase activity on a certain type of duplex DNA

In the search for proteins reactive to apurinic/apyrimidinic (AP) sites, it has been earlier found that proteins of human cell extracts formed the Schiff-base-dependent covalent adduct with an apparent molecular mass of 100kDa with a partial DNA duplex containing an AP site and 5′- and 3′-protruding ends (DDE-AP DNA). The adduct of such electrophoretic mobility was characteristic of only DDE-AP DNA (Ilina et al., Biochem. Biophys. Acta 1784 (2008) 1777–1785). The protein in this unusual adduct was identified as the Ku80 subunit of Ku antigen by peptide mass mapping based on MALDI-TOF MS data (Kosova et al., Biopolym. Cell 30 (2014) 42–46). Here we studied the interaction of Ku with DDE-AP DNA in details. Purified Ku (the Ku80 subunit) was shown to form the 100-kDa adduct highly specific for AP DNA with a certain length of protruding ends, base opposite the AP site and AP site location. Ku is capable of AP site cleavage in DDE-AP DNA unlike in analogous AP DNA with blunt ends. Ku cleaves AP sites via β-elimination and prefers apurinic sites over apyrimidinic ones. The AP site in DDE-DNA can be repaired in an apurinic/apyrimidinic endonuclease-independent manner via the successive action of Ku (cleavage of the AP site), tyrosyl-DNA phosphodiesterase 1 (removal of the 3ʹ-deoxyribose residue), polynucleotide kinase 3ʹ-phosphatase (removal of the 3ʹ-phosphate), DNA polymerase β (incorporation of dNMP), and DNA ligase (sealing the nick). These results provide a new insight into the role of Ku in the repair of AP sites.

DNA Repair Proteins XLF and XRCC4 Interact With Telomeric Proteins TRF1 And RAP1

Non‐homologous end‐joining (NHEJ) is the main repair pathway for processing double‐strand DNA breaks in Eukarya. In mammalian cells, NHEJ is mediated by a complex of proteins comprised of the Ku subunits (Ku70 and Ku86), Ligase IV, XRCC4, Artemis, DNA‐PKcs and XLF. Paradoxically, several of these NHEJ proteins are also found at telomeres, despite the fact that one of the main functions of the complex structure at telomeres is to prevent NHEJ proteins from recognizing the natural ends of chromosomes as DSBs. For instance, Ku is present at telomeres through interactions with resident telomeric proteins like TRF1 and TRF2, as is the case for DNA‐PKcs. Currently, the telomeric status of several NHEJ proteins is still unknown. Using protein fragment complementation assays, we have found interactions between two NHEJ proteins, XLF and XRCC4, with TRF1 and RAP1, two proteins present at telomeres. Co‐localization studies with TRF2 indicate that TRF1 interactions with XLF and XRCC4 occur at telomeres, while XLF‐RAP1 and XRCC4‐RAP1 interactions occur throughout the nucleus. Mutations that disrupt XRCC4‐XLF filament formation did not impair XRCC4 or XLF interaction with TRF1. Similarly, XRCC4 mutations in known phosphorylation sites for ATM and DNA‐PKcs did not impair XRCC4‐TRF1 interaction nor did they impair XRCC4 localization at telomeres. Overall, these results suggest that XRCC4 and XLF can localize at telomeres through direct interaction with telomeric resident proteins.Support or Funding InformationSupported by the UST/HCCS STEM Scholars Program (P031C110128‐12) from the US Department of Education and by the University of St Thomas Cullen Smith Chair of Biology

A novel cytoprotective function for the DNA repair protein Ku in regulating p53 mRNA translation andfunction.

Ku heterodimer is a DNA binding protein with a prominent role in DNA repair. Here, we investigate whether and how Ku impacts the DNA damage response by acting as a post-transcriptional regulator of gene expression. We show that Ku represses p53 protein synthesis and p53-mediated apoptosis by binding to a bulged stem-loop structure within the p53 5' UTR However, Ku-mediated translational repression of the p53 mRNA is relieved after genotoxic stress. The underlying mechanism involves Ku acetylation which disrupts Ku-p53 mRNA interactions. These results suggest that Ku-mediated repression of p53 mRNA translation constitutes a novel mechanism linking DNA repair and mRNA translation.

The Ku subunit of telomerase binds Sir4 to recruit telomerase to lengthen telomeres in S. cerevisiae.

In Saccharomyces cerevisiae and in humans, the telomerase RNA subunit is bound by Ku, a ring-shaped protein heterodimer best known for its function in DNA repair. Ku binding to yeast telomerase RNA promotes telomere lengthening and telomerase recruitment to telomeres, but how this is achieved remains unknown. Using telomere-length analysis and chromatin immunoprecipitation, we show that Sir4 - a previously identified Ku-binding protein that is a component of telomeric silent chromatin - is required for Ku-mediated telomere lengthening and telomerase recruitment. We also find that specifically tethering Sir4 directly to Ku-binding-defective telomerase RNA restores otherwise-shortened telomeres to wild-type length. These findings suggest that Sir4 is the telomere-bound target of Ku-mediated telomerase recruitment and provide one mechanism for how the Sir4-competing Rif1 and Rif2 proteins negatively regulate telomere length in yeast.

The Ku heterodimer: function in DNA repair and beyond.

Ku is an abundant, highly conserved DNA binding protein found in both prokaryotes and eukaryotes that plays essential roles in the maintenance of genome integrity. In eukaryotes, Ku is a heterodimer comprised of two subunits, Ku70 and Ku80, that is best characterized for its central role as the initial DNA end binding factor in the "classical" non-homologous end joining (C-NHEJ) pathway, the main DNA double-strand break (DSB) repair pathway in mammals. Ku binds double-stranded DNA ends with high affinity in a sequence-independent manner through a central ring formed by the intertwined strands of the Ku70 and Ku80 subunits. At the break, Ku directly and indirectly interacts with several C-NHEJ factors and processing enzymes, serving as the scaffold for the entire DNA repair complex. There is also evidence that Ku is involved in signaling to the DNA damage response (DDR) machinery to modulate the activation of cell cycle checkpoints and the activation of apoptosis. Interestingly, Ku is also associated with telomeres, where, paradoxically to its DNA end-joining functions, it protects the telomere ends from being recognized as DSBs, thereby preventing their recombination and degradation. Ku, together with the silent information regulator (Sir) complex is also required for transcriptional silencing through telomere position effect (TPE). How Ku associates with telomeres, whether it is through direct DNA binding, or through protein-protein interactions with other telomere bound factors remains to be determined. Ku is central to the protection of organisms through its participation in C-NHEJ to repair DSBs generated during V(D)J recombination, a process that is indispensable for the establishment of the immune response. Ku also functions to prevent tumorigenesis and senescence since Ku-deficient mice show increased cancer incidence and early onset of aging. Overall, Ku function is critical to the maintenance of genomic integrity and to proper cellular and organismal development.

7B7: a novel antibody directed against the Ku70/Ku80 heterodimer blocks invasion in pancreatic and lung cancer cells.

Development of more effective therapeutic strategies for cancers of high unmet need requires the continued discovery of disease-specific protein targets for therapeutic antibody targeting. In order to identify novel proteins associated with cancer cell invasion/metastasis, we present here an alternative to antibody targeting of cell surface proteins with an established role in invasion; our functional antibody screening approach involves the isolation and selection of MAbs that are primarily screened for their ability to inhibit tumour invasion. A clonal population of the Mia PaCa-2, a pancreatic ductal adenocarcinoma (PDAC) cell line, which displays a highly invasive phenotype, was used to generate MAbs with the objective of identifying membrane targets directly involved in cancer invasion. Selected MAb 7B7 can significantly reduce invasion in a dose-responsive manner in Mia PaCa-2 clone 3 and DLKP-M squamous lung carcinoma cells. Using immunoprecipitation and liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis, the target antigen of anti-invasive antibody, 7B7, was determined to be the heterodimeric Ku antigen, Ku70/80, a core protein composed of the Ku70 and Ku80 subunits which is involved in non-homologous end-joining (NHEJ) DNA repair. RNA interference-mediated knockdown of Ku70 and Ku80 resulted in a marked decrease in the invasive capacity of Mia PaCa-2 clone 3 and DLKP-M cells, indicating that Ku70/Ku80 is functionally involved in pancreatic and lung cancer invasion. Immunohistochemical analysis demonstrated Ku70/Ku80 immunoreactivity in 37 PDAC tumours, indicating that this heterodimer is highly expressed in this aggressive cancer type. This study demonstrates that a functional MAb screening approach coupled with immunoprecipitation/proteomic analyses can be successfully applied to identify functional anti-invasive MAbs and potential novel targets for therapeutic antibody targeting.

Deletion of Individual Ku Subunits in Mice Causes an NHEJ-Independent Phenotype Potentially by Altering Apurinic/Apyrimidinic Site Repair

Ku70 and Ku80 form a heterodimer called Ku that forms a holoenzyme with DNA dependent-protein kinase catalytic subunit (DNA-PKCS) to repair DNA double strand breaks (DSBs) through the nonhomologous end joining (NHEJ) pathway. As expected mutating these genes in mice caused a similar DSB repair-defective phenotype. However, ku70-/- cells and ku80-/- cells also appeared to have a defect in base excision repair (BER). BER corrects base lesions, apurinic/apyrimidinic (AP) sites and single stand breaks (SSBs) utilizing a variety of proteins including glycosylases, AP endonuclease 1 (APE1) and DNA Polymerase β (Pol β). In addition, deleting Ku70 was not equivalent to deleting Ku80 in cells and mice. Therefore, we hypothesized that free Ku70 (not bound to Ku80) and/or free Ku80 (not bound to Ku70) possessed activity that influenced BER. To further test this hypothesis we performed two general sets of experiments. The first set showed that deleting either Ku70 or Ku80 caused an NHEJ-independent defect. We found ku80-/- mice had a shorter life span than dna-pkcs-/- mice demonstrating a phenotype that was greater than deleting the holoenzyme. We also found Ku70-deletion induced a p53 response that reduced the level of small mutations in the brain suggesting defective BER. We further confirmed that Ku80-deletion impaired BER via a mechanism that was not epistatic to Pol β. The second set of experiments showed that free Ku70 and free Ku80 could influence BER. We observed that deletion of either Ku70 or Ku80, but not both, increased sensitivity of cells to CRT0044876 (CRT), an agent that interferes with APE1. In addition, free Ku70 and free Ku80 bound to AP sites and in the case of Ku70 inhibited APE1 activity. These observations support a novel role for free Ku70 and free Ku80 in altering BER.

Chemoproteomic Profiling Identifies Changes in DNA-PK as Markers of Early Dengue Virus Infection

Many cellular factors are regulated via mechanisms affecting protein conformation, localization, and function that may be undetected by most commonly used RNA- and protein-based profiling methods that monitor steady-state gene expression. Mass-spectrometry-based chemoproteomic profiling provides alternatives for interrogating changes in the functional properties of proteins that occur in response to biological stimuli, such as viral infection. Taking dengue virus 2 (DV2) infection as a model system, we utilized reactive ATP- and ADP-acyl phosphates as chemical proteomic probes to detect changes in host kinase function that occur within the first hour of infection. The DNA-dependent protein kinase (DNA-PK) was discovered as a host enzyme with significantly elevated probe labeling within 60 min of DV2 infection. Increased probe labeling was associated with increased DNA-PK activity in nuclear lysates and localization of DNA-PK in nucleoli. These effects on DNA-PK were found to require a postfusion step of DV2 entry and were recapitulated by transfection of cells with RNA corresponding to stem loop B of the DV2 5' untranslated region. Upon investigation of the potential downstream consequences of these phenomena, we detected a modest but significant reduction in the interferon response induced by DV2 in cells partially depleted of the Ku80 subunit of DNA-PK. These findings identify changes in DNA-PK localization and activity as very early markers of DV2 infection. More broadly, these results highlight the utility of chemoproteomic profiling as a tool to detect changes in protein function associated with different cell states and that may occur on very short time scales.