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Susan P Leesmiller - One of the best experts on this subject based on the ideXlab platform.
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dna requirements for interaction of the c terminal region of Ku80 with the dna dependent protein kinase catalytic subunit dna pkcs
2017Co-Authors: Sarvan Kumar Radhakrishnan, Susan P LeesmillerAbstract:Non-homologous end joining (NHEJ) is the major pathway for the repair of ionizing radiation induced DNA double strand breaks (DSBs) in human cells. Critical to NHEJ is the DNA-dependent interaction of the Ku70/80 heterodimer with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to form the DNA-PK holoenzyme. However, precisely how Ku recruits DNA-PKcs to DSBs ends to enhance its kinase activity has remained enigmatic, with contradictory findings reported in the literature. Here we address the role of the Ku80 C-terminal region (CTR) in the DNA-dependent interaction of Ku70/80 with DNA-PKcs using purified components and defined DNA structures. Our results show that the Ku80 CTR is required for interaction with DNA-PKcs on short segments of blunt ended 25bp dsDNA or 25bp dsDNA with a 15-base poly dA single stranded (ss) DNA extension, but this requirement is less stringent on longer dsDNA molecules (35bp blunt ended dsDNA) or 25bp duplex DNA with either a 15-base poly dT or poly dC ssDNA extension. Moreover, the DNA-PKcs-Ku complex preferentially forms on 25 bp DNA with a poly-pyrimidine ssDNA extension.Our work clarifies the role of the Ku80 CTR and dsDNA ends on the interaction of DNA-PKcs with Ku and provides key information to guide assembly and biology of NHEJ complexes.
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dna pk dependent phosphorylation of ku70 80 is not required for non homologous end joining
2005Co-Authors: Pauline Douglas, Katheryn Meek, Shikha Gupta, Nick Morrice, Susan P LeesmillerAbstract:The Ku70/80 heterodimer is a major player in non-homologous end joining and the repair of DNA double-strand breaks. Studies suggest that once bound to a DNA double-strand break, Ku recruits the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) to form the DNA-dependent protein kinase holoenzyme complex (DNA-PK). We previously identified four DNA-PK phosphorylation sites on the Ku70/80 heterodimer: serine 6 of Ku70, serine 577 and 580 and threonine 715 of Ku80. This raised the interesting possibility that DNA-PK-dependent phosphorylation of Ku could provide a mechanism for the regulation of non-homologous end joining. Here, using mass spectrometry and phosphospecific antibodies we confirm that these sites are phosphorylated in vitro by purified DNA-PK. However, we show that neither DNA-PK nor the related protein kinase ataxia-telangiectasia mutated (ATM) is required for phosphorylation of Ku at these sites in vivo. Furthermore, Ku containing serine/threonine to alanine mutations at these sites was fully able to complement the radiation sensitivity of Ku negative mammalian cells indicating that phosphorylation at these sites is not required for non-homologous end joining. Interestingly, both Ku70 and Ku80 were phosphorylated in cells treated with the protein phosphatase inhibitor okadaic acid under conditions known to inactivate protein phosphatase 2A-like protein phosphatases. Moreover, okadaic acid-induced phosphorylation of Ku80 was inhibited by nanomolar concentrations of the protein kinase inhibitor staurosporine. These results suggest that the phosphorylation of Ku70 and Ku80 is regulated by a protein phosphatase 2A-like protein phosphatase and a staurosporine sensitive protein kinase in vivo, but that DNA-PK-mediated phosphorylation of Ku is not required for DNA double-strand break repair.
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ku70 Ku80 and dna dependent protein kinase catalytic subunit modulate rag mediated cleavage implications for the enforcement of the 12 23 rule
2004Co-Authors: Dennis J Sawchuk, Susan P Leesmiller, Michel C Nussenzweig, Jorge Mansillasoto, Claudio Alarcon, Netai C Singha, Hanno Langen, Marco Bianchi, Patricia CortesAbstract:The 12/23 rule is a critical step for regulation of V(D)J recombination. To date, only the RAG proteins and high mobility group protein 1 or 2 have been implicated in 12/23 regulation. Through protein fractionation and biochemical experiments, we find that Ku70/Ku80 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) modulate RAG-mediated cleavage. Modulation of cleavage by Ku70/80 and DNA-PKcs results in preferential inhibition of 12/12 and 23/23 DNA cleavage, thus increasing 12/23 rule specificity. This observation indicates that DNA repair factors, Ku70/80 and DNA-PKcs, might be present upstream of the DNA cleavage events and not recruited downstream as is currently thought, assigning new nonrepair functions to the DNA-dependent protein kinase.
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dna dependent protein kinase phosphorylation sites in ku 70 80 heterodimer
1999Co-Authors: Doug W. Chan, Christian Veillette, Susan P LeesmillerAbstract:Ku antigen is composed of 70 and 82 kDa subunits (Ku70 and Ku80, respectively) that together bind with high affinity to ends of double-stranded DNA and other DNA structures in vitro. When bound to DNA, the Ku 70/80 heterodimer enhances the kinase activity of the catalytic subunit of the DNA-dependent protein kinase, DNA-PKcs. Ku and DNA-PKcs are required for V(D)J recombination and DNA double-strand break repair in vivo and may also play a role in regulation of transcription. Ku is phosphorylated by DNA-PKcs in vitro, and cells that lack DNA-PKcs are deficient in Ku phosphorylation in vitro, suggesting that Ku may be a physiological target for DNA-PK. Here we have identified the sites of DNA-PK phosphorylation in human Ku protein. We find that Ku70 is phosphorylated at a single serine residue, serine 6, located in the putative transcriptional activation domain, and Ku80 is phosphorylated at serines 577 and 580 and at threonine 715. Interestingly, none of the phosphorylation sites identified in Ku correspo...
Sakayu Shimizu - One of the best experts on this subject based on the ideXlab platform.
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gene targeting in the oil producing fungus mortierella alpina 1s 4 and construction of a strain producing a valuable polyunsaturated fatty acid
2015Co-Authors: Hiroshi Kikukawa, Eiji Sakuradani, Masato Nakatani, Akinori Ando, Tomoyo Okuda, Takaiku Sakamoto, Misa Ochiai, Sakayu ShimizuAbstract:To develop an efficient gene-targeting system in Mortierella alpina 1S-4, we identified the Ku80 gene encoding the Ku80 protein, which is involved in the nonhomologous end-joining pathway in genomic double-strand break (DSB) repair, and constructed Ku80 gene-disrupted strains via single-crossover homologous recombination. The ΔKu80 strain from M. alpina 1S-4 showed no negative effects on vegetative growth, formation of spores, and fatty acid productivity, and exhibited high sensitivity to methyl methanesulfonate, which causes DSBs. Dihomo-γ-linolenic acid (DGLA)-producing strains were constructed by disruption of the Δ5-desaturase gene, encoding a key enzyme of bioconversion of DGLA to ARA, using the ΔKu80 strain as a host strain. The significant improvement of gene-targeting efficiency was not observed by disruption of the Ku80 gene, but the construction of DGLA-producing strain by disruption of the Δ5-desaturase gene was succeeded using the ΔKu80 strain as a host strain. This report describes the first study on the identification and disruption of the Ku80 gene in zygomycetes and construction of a DGLA-producing transformant using a gene-targeting system in M. alpina 1S-4.
David J. Chen - One of the best experts on this subject based on the ideXlab platform.
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the Ku80 carboxy terminus stimulates joining and artemis mediated processing of dna ends
2009Co-Authors: Eric Weterings, Guido Keijzers, David J. Chen, Nicole S Verkaik, Bogdan I Florea, Shih Ya Wang, Laura Ortega, Naoya Uematsu, Dik C Van GentAbstract:Repair of DNA double-strand breaks (DSBs) is predominantly mediated by nonhomologous end joining (NHEJ) in mammalian cells. NHEJ requires binding of the Ku70-Ku80 heterodimer (Ku70/80) to the DNA ends and subsequent recruitment of the DNA-dependent protein kinase catalytic subunit (DNA-PKCS) and the XRCC4/ligase IV complex. Activation of the DNA-PKCS serine/threonine kinase requires an interaction with Ku70/80 and is essential for NHEJ-mediated DSB repair. In contrast to previous models, we found that the carboxy terminus of Ku80 is not absolutely required for the recruitment and activation of DNA-PKCS at DSBs, although cells that harbored a carboxy-terminal deletion in the Ku80 gene were sensitive to ionizing radiation and showed reduced end-joining capacity. More detailed analysis of this repair defect showed that DNA-PKCS autophosphorylation at Thr2647 was diminished, while Ser2056 was phosphorylated to normal levels. This resulted in severely reduced levels of Artemis nuclease activity in vivo and in vitro. We therefore conclude that the Ku80 carboxy terminus is important to support DNA-PKCS autophosphorylation at specific sites, which facilitates DNA end processing by the Artemis endonuclease and the subsequent joining reaction.
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solution structure of the c terminal domain of Ku80 suggests important sites for protein protein interactions
2004Co-Authors: Ziming Zhang, David J. Chen, Leticia Cano, Terry D Lee, Yuan ChenAbstract:The solution structure of Ku80 CTD from residue 566 to 732 has been solved in order to gain insights into the mechanisms of its interactions with other proteins. The structure reveals a topology similar to several common scaffolds for protein-protein interactions, in the absence of significant sequence similarity to these proteins. Conserved surface amino acid residues are clustered on two main surface areas, which are likely involved in mediating interactions between Ku80 and other proteins. The Ku70/Ku80 heterodimer has been shown to be involved in at least three processes, nonhomologous end joining, transcription, and telomere maintenance, and thus it needs to interact with different proteins involved in these different processes. The three-dimensional structure of the Ku80 C-terminal domain and the availability of NMR chemical shift assignments provide a basis for further investigation of the interactions between Ku80 and other proteins in these Ku-dependent cellular functions.
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the three dimensional structure of the c terminal dna binding domain of human ku70
2001Co-Authors: David J. Chen, Fanqing Chen, Ziming Zhang, Lingyang Zhu, Donghai Lin, Yuan ChenAbstract:The proteins Ku70 (69.8 kDa) and Ku80 (82.7 kDa) form a heterodimeric complex that is an essential component of the nonhomologous end joining DNA double-strand break repair pathway in mammalian cells. Interaction of Ku with DNA is central for the functions of Ku. Ku70, which is mainly responsible for the DNA binding activity of the Ku heterodimer, contains two DNA-binding domains. We have solved the solution structure of the Ku80-independent DNA-binding domain of Ku70 encompassing residues 536–609 using nuclear magnetic resonance spectroscopy. Residues 536–560 are highly flexible and have a random structure but form specific interactions with DNA. Residues 561–609 of Ku70 form a well defined structure with 3 α-helices and also interact with DNA. The three-dimensional structure indicates that all conserved hydrophobic residues are in the hydrophobic core and therefore may be important for structural integrity. Most of the conserved positively charged residues are likely to be critical for DNA recognition. The C-terminal DNA-binding domain of Ku70 contains a helix-extended strand-helix motif, which occurs in other nucleic acid-binding proteins and may represent a common nucleic acid binding motif.
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catalytic subunit of dna dependent protein kinase impact on lymphocyte development and tumorigenesis
1999Co-Authors: Akihiro Kurimasa, Honghai Ouyang, Li Jin Dong, Sa Wang, Xiaoling Li, Carlos Cordoncardo, David J. Chen, Gloria C LiAbstract:The DNA-dependent protein kinase (DNA-PK) consists of a heterodimer DNA-binding complex, Ku70 and Ku80, and a large catalytic subunit, DNA-PKcs. To examine the role of DNA-PKcs in lymphocyte development, radiation sensitivity, and tumorigenesis, we disrupted the mouse DNA-PKcs by homologous recombination. DNA-PKcs-null mice exhibit neither growth retardation nor a high frequency of T cell lymphoma development, but show severe immunodeficiency and radiation hypersensitivity. In contrast to the Ku70−/− and Ku80−/− phenotype, DNA-PKcs-null mice are blocked for V(D)J coding but not for signal-end joint formation. Furthermore, inactivation of DNA-PKcs leads to hyperplasia and dysplasia of the intestinal mucosa and production of aberrant crypt foci, suggesting a novel role of DNA-PKcs in tumor suppression.
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A central region of Ku80 mediates interaction with Ku70 in vivo
1998Co-Authors: Robert B. Cary, Fanqing Chen, Zhiyuan Shen, David J. ChenAbstract:Ku, the DNA binding component of DNA-dependent protein kinase (DNA-PK), is a heterodimer composed of 70 and 86 kDa subunits, known as Ku70 and Ku80 respectively . Defects in DNA-PK subunits have been shown to result in a reduced capacity to repair DNA double-strand breaks. Assembly of the Ku heterodimer is required to obtain DNA end binding activity and association of the DNA-PK catalytic subunit. The regions of the Ku subunits responsible for heterodimerization have not been clearly defined in vivo . A previous study has suggested that the C-terminus of Ku80 is required for interaction with Ku70. Here we examine Ku subunit interaction using N- and C-terminal Ku80 deletions in a GAL4-based two-hybrid system and an independent mammalian in vivo system. Our two-hybrid study suggests that the central region of Ku80, not its C-terminus, is capable of mediating interaction with Ku70. To determine if this region mediates interaction with Ku70 in mammalian cells we transfected xrs-6 cells, which lack endogenous Ku80, with epitope-tagged Ku80 deletions carrying a nuclear localization signal. Immunoprecipitation from transfected cell extracts revealed that the central domain identified by the GAL4 two-hybrid studies stabilizes and co-immunoprecipitates with endogenous xrs-6 Ku70. The central interaction domain maps to the internally deleted regions of Ku80 in the mutant cell lines XR-V9B and XR-V15B. These findings indicate that the internally deleted Ku80 mutations carried in these cell lines are incapable of heterodimerization with Ku70.
Frederick W Alt - One of the best experts on this subject based on the ideXlab platform.
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synthetic lethality between murine dna repair factors xlf and dna pkcs is rescued by inactivation of ku70
2017Co-Authors: Mengtan Xing, Frederick W Alt, Magnar Bjoras, Jeremy A Daniel, Valentyn OksenychAbstract:DNA double-strand breaks (DSBs) are recognized and repaired by the Classical Non-Homologous End-Joining (C-NHEJ) and Homologous Recombination pathways. C-NHEJ includes the core Ku70 and Ku80 (or Ku86) heterodimer that binds DSBs and thus promotes recruitment of accessory downstream NHEJ factors XLF, PAXX, DNA-PKcs, Artemis and other core subunits, XRCC4 and DNA Ligase 4 (Lig4). In the absence of core C-NHEJ factors, DNA repair can be performed by Alternative End-Joining, which likely depends on DNA Ligase 1 and DNA Ligase 3. Genetic inactivation of C-NHEJ factors, such as Ku70, Ku80, XLF, PAXX and DNA-PKcs results in viable mice showing increased levels of genomic instability and sensitivity to DSBs. Knockouts of XRCC4 or Lig4, on the other hand, as well as combined inactivation of XLF and DNA-PKcs, or XLF and PAXX, result in late embryonic lethality in mice, which in most cases correlate with severe apoptosis in the central nervous system. Here, we demonstrate that inactivation of the Ku70 gene rescues the synthetic lethality between XLF and DNA-PKcs, resulting in triple knockout mice that are indistinguishable from Ku70-deficient littermates by size or levels of genomic instability. Moreover, we find that combined inactivation of Ku70 and XLF results in viable mice. Together, these findings suggest that Ku70 is epistatic with XLF and DNA-PKcs and support a model in which inactivation of Ku70 allows DNA lesions to become accessible to alternative DNA repair pathways.
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defective embryonic neurogenesis in ku deficient but not dna dependent protein kinase catalytic subunit deficient mice
2000Co-Authors: Joann Sekiguchi, Paul Hasty, Yijie Gao, Karen M Frank, Pieter Dikkes, David J P Ferguson, Jerold Chun, Frederick W AltAbstract:Mammalian nonhomologous DNA end joining employs Ku70, Ku80, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4, and DNA ligase IV (Lig4). Herein, we show that Ku70 and Ku80 deficiency but not DNA-PKcs deficiency results in dramatically increased death of developing embryonic neurons in mice. The Ku-deficient phenotype is qualitatively similar to, but less severe than, that associated with XRCC4 and Lig4 deficiency. The lack of a neuronal death phenotype in DNA-PKcs-deficient embryos and the milder phenotype of Ku-deficient versus XRCC4- or Lig4-deficient embryos correlate with relative leakiness of residual end joining in these mutant backgrounds as assayed by a V(D)J recombination end joining assay. We conclude that normal development of the nervous system depends on the four evolutionarily conserved nonhomologous DNA end joining factors.
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a targeted dna pkcs null mutation reveals dna pk independent functions for ku in v d j recombination
1998Co-Authors: Yijie Gao, David T Weaver, Jayanta Chaudhuri, Chengming Zhu, Laurie Davidson, Frederick W AltAbstract:The DNA-dependent protein kinase (DNA-PK) consists of Ku70, Ku80, and a large catalytic subunit, DNA-PKcs. Targeted inactivation of the Ku70 or Ku80 genes results in elevated ionizing radiation (IR) sensitivity and inability to perform both V(D)J coding-end and signal (RS)-end joining in cells, with severe growth retardation plus immunodeficiency in mice. In contrast, we now demonstrate that DNA-PKcs-null mice generated by gene-targeted mutation, while also severely immunodeficient, exhibit no growth retardation. Furthermore, DNA-PKcs-null cells are blocked for V(D)J coding-end joining, but retain normal RS-end joining. Finally, while DNA-PK-null fibroblasts exhibited increased IR sensitivity, DNA-PKcs-deficient ES cells did not. We conclude that Ku70 and Ku80 may have functions in V(D)J recombination and DNA repair that are independent of DNA-PKcs.
Manabu Koike - One of the best experts on this subject based on the ideXlab platform.
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accumulation of Ku80 proteins at dna double strand breaks in living cells
2008Co-Authors: Manabu Koike, Aki KoikeAbstract: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 alpha/beta, the DNA-binding, and Ku70-binding domains, seem to necessary for the accumulation at or recognition of DSBs in the early stage after irradiation.
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The Ku70-binding site of Ku80 is required for the stabilization of Ku70 in the cytoplasm, for the nuclear translocation of Ku80, and for Ku80-dependent DNA repair.
2005Co-Authors: Manabu Koike, Aki KoikeAbstract:Ku plays a key role in multiple nuclear processes, e.g., DNA repair, transcription regulation, and replication. It is believed that heterodimerization between Ku70 and Ku80 is essential for Ku-dependent DNA repair, although its role is poorly understood. We previously identified the Ku70-binding site of Ku80. In this study, to understand the role of heterodimerization in the function of Ku, we generated and/or analyzed cell lines stably expressing the EGFP-tagged-wild-type human Ku80, its Ku70-binding mutant, its NLS-dysfunctional mutant, or its double mutant in Ku80-deficient cells. Our results show that the Ku70-binding site of Ku80 is required for the stabilization of Ku70 in the cytoplasm and for the nuclear translocation of Ku80 through its heterodimerization with Ku70. In addition, our results suggest that the nuclear translocation of Ku80 through the Ku70-binding site as well as through the NLS of Ku80 play, at least in part, a role in Ku80-dependent DNA repair. Furthermore, our results suggest the possibility that Ku80 has a DNA DSB repair function independent of Ku70 in the nuclei, in addition to that dependent on Ku70.
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Dimerization, translocation and localization of Ku70 and Ku80 proteins.
2002Co-Authors: Manabu KoikeAbstract:The Ku protein is a complex of two subunits, Ku70 and Ku80, and was originally identified as an autoantigen recognized by the sera of patients with autoimmune diseases. The Ku protein plays a key role in multiple nuclear processes, e.g., DNA repair, chromosome maintenance, transcription regulation, and V(D)J recombination. The mechanism underlying the regulation of all the diverse functions of Ku is still unclear, although it seems that Ku is a multifunctional protein that works in nuclei. On the other hand, several studies have reported cytoplasmic or cell surface localization of Ku in various cell types. To clarify the fundamental characteristics of Ku, we have examined the expression, heterodimerization, subcellular localization, chromosome location, and molecular mechanisms of the nuclear transport of Ku70 and Ku80. The mechanism that regulates for nuclear localization of Ku70 and Ku80 appears to play, at least in part, a key role in regulating the physiological function of Ku in vivo.
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Ku80 can translocate to the nucleus independent of the translocation of ku70 using its own nuclear localization signal
1999Co-Authors: Manabu Koike, Takashi Miyasaka, Togo Ikuta, Tadahiro ShiomiAbstract:Ku antigen is a complex of Ku70 and Ku80 subunits and plays an important role in not only DNA double-strand breaks (DSB) repair and V(D)J recombination, but also in growth regulation. Ku is generally believed to always form and function as heterodimers on the basis of in vitro observations. Here we demonstrate that the localization of Ku80 does not completely coincide with that of Ku70. Ku70 and Ku80 were colocalized in the nucleus in the interphase but not in the late telophase/early G1 phase of the cell cycle. Since the in vivo function of Ku might be partially regulated by the control of its transport, we attempted to investigate the molecular mechanisms underlying the nuclear translocation of Ku. The nuclear translocation of Ku80 started during the late telophase/early G1 phase after the nuclear envelope was formed and this was preceded by the nuclear translocation of Ku70. Furthermore, we found that the Ku80 protein was transported to the nucleus without heterodimerization with Ku70. To understand in detail the mechanism of transport of Ku80, we attempted to identify the nuclear localization signal (NLS) of Ku80 and defined to a region spanning nine amino acid residues (positions 561 - 569). The Ku80 NLS was demonstrated to be mediated to the nuclear rim by two components of PTAC58 and PTAC97. All these findings support the idea that Ku80 can translocate to the nucleus using its own NLS independent of the translocation of Ku70.
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Differential subcellular localization of DNA-dependent protein kinase components Ku and DNA-PKcs during mitosis
1999Co-Authors: Manabu Koike, Takeo Awaji, Masakazu Kataoka, Gozoh Tsujimoto, Tonja Kartasova, Aki Koike, Tadahiro ShiomiAbstract:The Ku protein is a complex of two subunits, Ku70 and Ku80. Ku plays an important role in DNA-PKcs-dependent double-strand break repair and V(D)J recombination, and in growth regulation, which is DNA-PKcs-independent. We studied the expression and the subcellular localization of Ku and DNA-PKcs throughout the cell cycle in several established human cell lines. Using immunofluorescence analysis and confocal laser scanning microscopy, we detected Ku70 and Ku80 in the nuclei in interphase cells. In mitotic cells (1) most of Ku protein was found diffused in the cytoplasm, (2) a fraction was detected at the periphery of condensed chromosomes, (3) no Ku protein was present in the chromosome interior. Association of Ku with isolated chromosomes was also observed. On the other hand, DNA-PKcs was detected in the nucleus in interphase cells and not at the periphery of condensed chromosomes during mitosis. Using indirect immunoprecipitation, we found that throughout the cell cycle, Ku70 and Ku80 were present as heterodimers, some in complex with DNA-PKcs. Our findings suggest that the localization of Ku at the periphery of metaphase chromosomes might be imperative for a novel function of Ku in the G(2)/M phase, which does not require DNA-PKcs.
