Статті в журналах: "KU70 gene"

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Ouyang, Honghai, Andre Nussenzweig, Akihiro Kurimasa, Vera da Costa Soares, Xiaoling Li, Carlos Cordon-Cardo, Wen-hui Li, et al. "Ku70 Is Required for DNA Repair but Not for T Cell Antigen Receptor Gene Recombination In Vivo." Journal of Experimental Medicine 186, no. 6 (September 15, 1997): 921–29. http://dx.doi.org/10.1084/jem.186.6.921.

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Ku is a complex of two proteins, Ku70 and Ku80, and functions as a heterodimer to bind DNA double-strand breaks (DSB) and activate DNA-dependent protein kinase. The role of the Ku70 subunit in DNA DSB repair, hypersensitivity to ionizing radiation, and V(D)J recombination was examined in mice that lack Ku70 (Ku70−/−). Like Ku80−/− mice, Ku70−/− mice showed a profound deficiency in DNA DSB repair and were proportional dwarfs. Surprisingly, in contrast to Ku80−/− mice in which both T and B lymphocyte development were arrested at an early stage, lack of Ku70 was compatible with T cell receptor gene recombination and the development of mature CD4+CD8− and CD4−CD8+ T cells. Our data shows, for the first time, that Ku70 plays an essential role in DNA DSB repair, but is not required for TCR V(D)J recombination. These results suggest that distinct but overlapping repair pathways may mediate DNA DSB repair and V(D)J recombination.

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Li, Han, Hannes Vogel, Valerie B. Holcomb, Yansong Gu, and Paul Hasty. "Deletion of Ku70, Ku80, or Both Causes Early Aging without Substantially Increased Cancer." Molecular and Cellular Biology 27, no. 23 (September 17, 2007): 8205–14. http://dx.doi.org/10.1128/mcb.00785-07.

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ABSTRACT Ku70 forms a heterodimer with Ku80, called Ku, that is critical for repairing DNA double-stand breaks by nonhomologous end joining and for maintaining telomeres. Mice with either gene mutated exhibit similar phenotypes that include increased sensitivity to ionizing radiation and severe combined immunodeficiency. However, there are also differences in the reported phenotypes. For example, only Ku70 mutants are reported to exhibit a high incidence of thymic lymphomas while only Ku80 mutants are reported to exhibit early aging with very low cancer levels. There are two explanations for these differences. First, either Ku70 or Ku80 functions outside the Ku heterodimer such that deletion of one is not identical to deletion of the other. Second, divergent genetic backgrounds or environments influence the phenotype. To distinguish between these possibilities, the Ku70 and Ku80 mutations were crossed together to generate Ku70, Ku80, and double-mutant mice in the same genetic background raised in the same environment. We show that these three cohorts have similar phenotypes that most resemble the previous report for Ku80 mutant mice, i.e., early aging without substantially increased cancer levels. Thus, our observations suggest that the Ku heterodimer is important for longevity assurance in mice since divergent genetic backgrounds and/or environments likely account for these previously reported differences.

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Sucharov, Carmen C., Steve M. Helmke, Stephen J. Langer, M. Benjamin Perryman, Michael Bristow та Leslie Leinwand. "The Ku Protein Complex Interacts with YY1, Is Up-Regulated in Human Heart Failure, and Represses α Myosin Heavy-Chain Gene Expression". Molecular and Cellular Biology 24, № 19 (1 жовтня 2004): 8705–15. http://dx.doi.org/10.1128/mcb.24.19.8705-8715.2004.

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ABSTRACT Human heart failure is accompanied by repression of genes such as α myosin heavy chain (αMyHC) and SERCA2A and the induction of fetal genes such as βMyHC and atrial natriuretic factor. It seems likely that changes in MyHC isoforms contribute to the poor contractility seen in heart failure, because small changes in isoform composition can have a major effect on the contractility of cardiac myocytes and the heart. Our laboratory has recently shown that YY1 protein levels are increased in human heart failure and that YY1 represses the activity of the human αMyHC promoter. We have now identified a region of the αMyHC promoter that binds a factor whose expression is increased sixfold in failing human hearts. Through peptide mass spectrometry, we identified this binding activity to be a heterodimer of Ku70 and Ku80. Expression of Ku represses the human αMyHC promoter in neonatal rat ventricular myocytes. Moreover, overexpression of Ku70/80 decreases αMyHC mRNA expression and increases skeletal α-actin. Interestingly, YY1 interacts with Ku70 and Ku80 in HeLa cells. Together, YY1, Ku70, and Ku80 repress the αMyHC promoter to an extent that is greater than that with YY1 or Ku70/80 alone. Our results suggest that Ku is an important factor in the repression of the human αMyHC promoter during heart failure.

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Weterings, Eric, Nicole S. Verkaik, Guido Keijzers, Bogdan I. Florea, Shih-Ya Wang, Laura G. Ortega, Naoya Uematsu, David J. Chen, and Dik C. van Gent. "The Ku80 Carboxy Terminus Stimulates Joining and Artemis-Mediated Processing of DNA Ends." Molecular and Cellular Biology 29, no. 5 (December 22, 2008): 1134–42. http://dx.doi.org/10.1128/mcb.00971-08.

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ABSTRACT 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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Ghonim, Mohamed Ahmed, Kusma Pyakurel, Hanh Luu, Samuel Okpechi, Jihang Ju, and Hamid Boulares. "The catalytic subunit of DNA-PK has a unique function in inflammation independently of Ku70 and DNA repair: a new opportunity to target the enzyme without interfering with DNA repair." Journal of Immunology 200, no. 1_Supplement (May 1, 2018): 173.18. http://dx.doi.org/10.4049/jimmunol.200.supp.173.18.

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Abstract Our laboratory demonstrated a critical role for DNA-dependent protein kinase (DNA-PK) in asthma pathogenesis via modulating the pertinent immune responses. DNA-PK is DNA repair enzyme composed of a catalytic subunit (DNA-PKcs) and two DNA-binding subunits (Ku70 and Ku80). Human cells express high levels of DNA-PK, surprisingly such high levels do not confer increased ability to repair DNA damage. Here we show that the role of DNA-PK in promoting inflammatory responses is independent of its function in the DNA repair. We examined the effect(s) of partial depletion of Ku70 on Ovalbumin (OVA) induced lung inflammation in mouse model of the disease. Of note, depletion of Ku70 by gene heterozygosity causes deficiency in DNA-PK-dependent DNA repair. Unlike the protective effects provided by DNA-PKcs gene heterozygosity, Ku70 heterozygosity did not alter the OVA-induced eosinophilia, mucus hypersecretion, Th2 cytokines production, or OVA-specific IgE upon OVA challenge. Interestingly, Ku70 heterozygosity enhanced methacholine-induced AHR over that of WT mice. Using a cell culture system, we demonstrate that while IL-4 and TNF-α are potent inducers of DNA-PKcs, such activation did not coincide with any detectable DNA damage or repair responses. Remarkably, while Ku70−/− blocked DNA-PKcs autophosphorylation in response to the DNA damage agent, etoposide, it did not affect the kinase in response to TNFα. Our findings suggest that the mechanism by which DNA-PK functions in inflammation is completely unrelated to its role in DNA repair, thus, unraveling a completely novel function for the kinase. More importantly, this provides a window of opportunity to target DNA-PK in inflammatory diseases without interfering with DNA repair processes.

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6

Fu, Chengcheng, Peishuai Chen, Wu Depei, and Zixing Chen. "Study of Expression of Ku70 and NHEJ in CML Cells." Blood 112, no. 11 (November 16, 2008): 3223. http://dx.doi.org/10.1182/blood.v112.11.3223.3223.

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Abstract Objective FThe DNA double strand breaks (DSB) in mammalian cells are predominantly repaired by a process called non-homologous DNA end joining (NHEJ). Ku70 played a pivotal role in NHEJ pathway. As a common hematological malignant disease with unique chromosomal translocation t (9;22), chronic myeloid leukemia (CML) can be considered as a paradigm for neoplasias that evolve through a multi-step process. As we reported before, protein Ku70 expressed significantly higher in CML than in normal BM cells. Meanwhile, its expression level in blast phase was markedly higher than that in chronic phase. The present study furthermore aims to investigate the expression of the gene Ku70 in CML cells at different clinical stage and reveal the correlation among the expression of the gene Ku70, the protein Ku70 and BCR-ABL in cells of CML. The NHEJ efficiency to repair DSB in CML was also investigated. Methods: Bone marrow cells were collected from 24 cases of normal adults and 27 cases of de novo diagnosed CML patients. 15 CML patients were in chronic phase and 12 in blast phase. The expression of gene Ku70 was detected by RT-PCR and RQ-RT-PCR. The fusion gene BCR-ABL was detected by TaqMan probe Real Time PCR, using ABL as the internal control. Nucleic extracted proteins were used to determine NHEJ efficiency by an in vitro end-ligation system. Results: The mean level of NHEJ activity of normal BM cells and CML cells were 18.6±13.1% vs 24.8±14.9%, .024. The gene Ku 70 expression in normal BM cells and CML cells were 31.08±8.41 vs 544.63±1185.71 copies/10,000 β-Actin copies, P=0.039. gene Ku 70 expressed significantly higher in blast phase (1103.31±1645.62 copies/10,000 β-Actin copies, P<0.01). There were positive correlations of BCR-ABL when compared with the expression of the gene Ku70 (r=0.573 P=0.002) and with the protein Ku70 (r=0.705 P<0.001) in CML cells. There was also significantly correlations between the expression of the gene Ku70 and the protein Ku70 (r=0. 808, P<0.001). Conclusions: The gene and protein of Ku70 were expressed significantly higher in CML than in normal BM cells with NHEJ activity was also enhanced in CML cells. Meanwhile, Ku70 expression level in acute phase was markedly higher than that in chronic phase. There were significantly correlations among the expression of fusion gene BCR-ABL, the gene Ku70 and the protein Ku70 in cells of CML This Study illustrates DNA instability in CML cells which was mainly damaged by DSB, and this kind of DNA damage was repaired by NHEJ pathway predominantly. NHEJ pathway plays an important role in disease progression of CML.

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7

Gao, Chao, Guangxu Jin, Elizabeth Forbes, Lingegowda S. Mangala, Yingmei Wang, Cristian Rodriguez-Aguayo, Paola Amero, et al. "Inactivating Mutations of the IK Gene Weaken Ku80/Ku70-Mediated DNA Repair and Sensitize Endometrial Cancer to Chemotherapy." Cancers 13, no. 10 (May 20, 2021): 2487. http://dx.doi.org/10.3390/cancers13102487.

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IK is a mitotic factor that promotes cell cycle progression. Our previous investigation of 271 endometrial cancer (EC) samples from the Cancer Genome Atlas (TCGA) dataset showed IK somatic mutations were enriched in a cluster of patients with high-grade and high-stage cancers, and this group had longer survival. This study provides insight into how IK somatic mutations contribute to EC pathophysiology. We analyzed the somatic mutational landscape of IK gene in 547 EC patients using expanded TCGA dataset. Co-immunoprecipitation and mass spectrometry were used to identify protein interactions. In vitro and in vivo experiments were used to evaluate IK’s role in EC. The patients with IK-inactivating mutations had longer survival during 10-year follow-up. Frameshift and stop-gain were common mutations and were associated with decreased IK expression. IK knockdown led to enrichment of G2/M phase cells, inactivation of DNA repair signaling mediated by heterodimerization of Ku80 and Ku70, and sensitization of EC cells to cisplatin treatment. IK/Ku80 mutations were accompanied by higher mutation rates and associated with significantly better overall survival. Inactivating mutations of IK gene and loss of IK protein expression were associated with weakened Ku80/Ku70-mediated DNA repair, increased mutation burden, and better response to chemotherapy in patients with EC.

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Imamichi, Tomozumi, Xing Zhang, Ming Zhou, Richard Lempicki, Michael Baseler, Timothy Veenstra, Howard Young, and H. Clifford Lane. "Ku70 is a novel cytosolic DNA sensor that induces a Type-III rather than Type-I IFN via activation of IRF-1 and IRF-7. (116.11)." Journal of Immunology 186, no. 1_Supplement (April 1, 2011): 116.11. http://dx.doi.org/10.4049/jimmunol.186.supp.116.11.

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Abstract Recent studies have demonstrated that foreign cytoplasmic DNA is detected by cytosolic DNA sensors (DAI, AIM-2, LRRFIP1, RNA polymerase III, or IFI16) that induce Type-I IFN or IL-1β. We have previously found that HEK293 cell transfected with DNA induces a Type-III IFN (IFN-λ1) rather than Type-I IFN. In this study, utilizing a pull down assay followed by mass spectrometry, we identified the DNA sensor protein that specifically induces IFN-λ1. Changes in expression level of gene and protein were evaluated by real time RT-PCR and Western blot. Transfection of not only plasmid DNA but also PCR amplified DNA and bacteria DNA induced IFN-λ1 activation in cell lines, primary macrophages and dendritic cells. DNA virus (HSV-2) infection also induced IFN-λ1 production. The pull down assay using DNA-beads resulted in that DNA-binding proteins in cytosole were Ku70 and Ku80, a DNA repair protein, as playing potentially important role. Knockdown of Ku70, but not Ku80, by siRNA led to 90% and 70% reduction in the DNA- and the virus-mediated IFN-λ1 induction, respectively. Over expression of Ku70 induced activation of the IFN-λ1 promoter. DNA binding assay using NFκB-, ISRE- or PRDI-beads with nuclear extract from DNA transfected cells indicated that the DNA-mediated IFN-λ1 induction was associated with the activation of IRF-1 and IRF-7 rather than IRF-3 and NFκB. Taken together; these data demonstrated that Ku70 exerts a novel cytosolic DNA sensor that specifically activates IFN-λ1.

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Koh, Chong Mei, Yanbin Liu, Moehninsi, Minge Du, and Lianghui Ji. "Molecular characterization of KU70 and KU80 homologues and exploitation of a KU70-deficient mutant for improving gene deletion frequency in Rhodosporidium toruloides." BMC Microbiology 14, no. 1 (2014): 50. http://dx.doi.org/10.1186/1471-2180-14-50.

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Qing, Yulan, Zhengqi Wang, Shigemi Matsuyama, Kevin D. Bunting, and Stanton L. Gerson. "Rescue of the HSC Maintenance Defects in Ku70-Deficient Mice by Overexpression of Bcl2 Reveals a Novel Role of Bcl2 in HSC." Blood 120, no. 21 (November 16, 2012): 1235. http://dx.doi.org/10.1182/blood.v120.21.1235.1235.

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Abstract Abstract 1235 Ku70 is a key component of the non-homologous end joining (NHEJ) pathway; Ku70-deficient mice are hypersensitive to radiation and show a leaky SCID phenotype. We find that HSC from Ku70-deficient mice are severely impaired in maintenance, defective in self-renewal, competitive repopulation and BM hematopoietic niche occupancy. HSCs from Ku70-deficient mice are not prone to spontaneous apoptosis, however, they display more active proliferation, and fewer cells in a quiescent state than HSC obtained from WT mice. Further, gene expression profiling showed that multiple HSC quiescence- related genes such as c-MPL and p57 were significantly downregulated in Ku70-deficient HSCs. These data suggested that loss of quiescence results in the dramatic defect in the maintenance of Ku70-deficient HSC. Bcl2 plays important roles in both anti-apoptosis and anti-proliferation, while the anti-apoptosis function of Bcl2 has been extensively studied and well established in various settings, including HSCs, the anti-proliferation function of Bcl2 in HSC has yet to be investigated. To exam whether overexpression of Bcl2 can rescue the HSC defect in Ku70-deficient mice, H2K-Bcl2 transgenic/Ku70-deficient double mutant mice were generated. Though overexpression of Bcl2 does not rescue the SCID phenotype in Ku70-deficient mice, overexpression of Bcl2 in Ku70-deficient HSCs almost completely rescued the impaired HSC quiescence, repopulation and BM hematopoietic niche occupancy capacities. At the transcriptional level, overexpression of Bcl2 restored the expression of c-MPL and p57 comparable to WT levels. However, additional deletion of Bax, a pro-apoptosis protein in the Bcl2 family, in Ku70-deficient mice could not rescue the HSC repopulation defect, suggesting that apoptosis is not the main mediator for the impaired HSC maintenance in Ku70-deficient mice. Together, our data indicate that the HSC maintenance defect of Ku70-deficient mice is due to the loss of HSC quiescence populations, whereas overexpression of Bcl2 rescues the HSC defect in Ku70-deficient mice by restoration of quiescence. Our study uncovers a novel role of Bcl2 in hematopoietic stem cell quiescence regulation. Disclosures: No relevant conflicts of interest to declare.

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Lobbardi, Riadh, Jordan Pinder, Barbara Martinez-Pastor, Jessica S. Blackburn, Nouran Abdelfattah, Deborah Toiber, Manon De Waard, et al. "Thymocyte Selection-Associated High-Mobility Group Box Protein (TOX) Induces Genomic Instability in T-Cell Acute Lymphoblastic Leukemia." Blood 124, no. 21 (December 6, 2014): 475. http://dx.doi.org/10.1182/blood.v124.21.475.475.

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Abstract MYC and NOTCH are major oncogenic drivers in T-cell Acute Lymphoblastic Leukemia (T-ALL), yet additional collaborating genetic lesions likely collaborate to induce frank malignancy. To identify these factors, a large-scale transgenic screen was completed where 38 amplified and over-expressed genes found in human T-ALL were assessed for accelerating leukemia onset in the zebrafish transgenic model. From this analysis, Thymocyte selection-associated homeobox protein (TOX) synergized with both MYC and NOTCH to induce T-ALL. TOX is dynamically regulated in T cell development with peak expression occurring when thymocytes are actively undergoing T cell receptor (TCR) recombination. TOX is best known for regulating the specification of the mature CD4+ T cells. Despite TOX being genomically amplified in a subset of human and mouse T-ALL and being overexpressed in 100% of human T-ALL, a role for TOX in regulating leukemogenesis has not been reported. Characterization of zebrafish T-ALLs revealed that TOX expands the overall number of malignant T-ALL clones and promoted genomic instability as assessed by changes in DNA content. To identify TOX binding partners, antibody immunoprecipitation studies were performed followed by Tandem Mass Spectrometry. TOX was found to interact with KU70/KU80 but not other DNA repair enzymes including LigaseIV, DNA-PKC, or XRCC4. These results were verified by Western blot analysis and reciprocal immunoprecipitation studies using antibodies specific to KU70/KU80 both in the absence and presence of DNAseI treatment. Given that TOX elevated genomic instability in the zebrafish model and bound specifically to KU70/KU80 – the initiating factors required for Non-Homologous End Joining (NHEJ) repair - we hypothesized that TOX is a negative regulator of double-strand break repair. Fluorescent repair assays were completed in 3T3 fibroblasts and confirmed that TOX inhibits Non-Homologous End Joining (NHEJ). Both the nuclear localization signal and HMG-box were required for the ability of TOX to inhibit double-strand break repair. Dynamic real-time imaging studies confirmed that TOX suppresses recruitment of fluorescent-tagged KU70 to DNA breaks. Importantly, TOX loss of function increased NHEJ in human T-ALL cells and reduced time to DNA repair as assessed by fluorescent Traffic Light Reporter assays and quantitative assessment of 53BP1 and γH2A.X foci resolution following irradiation. Given the prominent role TOX has in T cell development and its coordinated regulation during active TCRβ and TCRα rearrangement, it is likely that the normal function of TOX is to transiently suppress the NHEJ pathway during Recombination-Activating Gene (RAG)-mediated recombination. Prolonging the time to DNA repair would likely facilitate long-range repair across VDJ segments. In the setting of T-ALL, TOX is aberrantly re-activated, thereby suppressing KU70/KU80 function to promote genomic instability and ultimately elevating rates at which acquired mutations and rearrangements are amassed in developing pre-malignant T cells. Disclosures No relevant conflicts of interest to declare.

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Takahashi, Tadashi, Masahiro Ogawa, and Yasuji Koyama. "Analysis of the Functions of Recombination-Related Genes in the Generation of Large Chromosomal Deletions by Loop-Out Recombination in Aspergillus oryzae." Eukaryotic Cell 11, no. 4 (January 27, 2012): 507–17. http://dx.doi.org/10.1128/ec.05208-11.

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ABSTRACT Loop-out-type recombination is a type of intrachromosomal recombination followed by the excision of a chromosomal region. The detailed mechanism underlying this recombination and the genes involved in loop-out recombination remain unknown. In the present study, we investigated the functions of ku70 , ligD , rad52 , rad54 , and rdh54 in the construction of large chromosomal deletions via loop-out recombination and the effect of the position of the targeted chromosomal region on the efficiency of loop-out recombination in Aspergillus oryzae . The efficiency of generation of large chromosomal deletions in the near-telomeric region of chromosome 3, including the aflatoxin gene cluster, was compared with that in the near-centromeric region of chromosome 8, including the tannase gene. In the Δ ku70 and Δ ku70-rdh54 strains, only precise loop-out recombination occurred in the near-telomeric region. In contrast, in the Δ ligD , Δ ku70-rad52 , and Δ ku70-rad54 strains, unintended chromosomal deletions by illegitimate loop-out recombination occurred in the near-telomeric region. In addition, large chromosomal deletions via loop-out recombination were efficiently achieved in the near-telomeric region, but barely achieved in the near-centromeric region, in the Δ ku70 strain. Induction of DNA double-strand breaks by I-SceI endonuclease facilitated large chromosomal deletions in the near-centromeric region. These results indicate that ligD , rad52 , and rad54 play a role in the generation of large chromosomal deletions via precise loop-out-type recombination in the near-telomeric region and that loop-out recombination between distant sites is restricted in the near-centromeric region by chromosomal structure.

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He, Yi, Qingpei Liu, Yanchun Shao, and Fusheng Chen. "ku70 and ku80 null mutants improve the gene targeting frequency in Monascus ruber M7." Applied Microbiology and Biotechnology 97, no. 11 (April 2, 2013): 4965–76. http://dx.doi.org/10.1007/s00253-013-4851-8.

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14

Belyashova, A., A. Golanov, G. Pavlova, E. Savchenko, N. Antipina, D. Panteleev, D. Shamadykova, G. Smirnov, and A. Nikolaeva. "P11.48 Dose-dependent effects of radiation exposure on the cell culture of glioblastoma G01, obtained from the patient with a long-term survival." Neuro-Oncology 21, Supplement_3 (August 2019): iii54. http://dx.doi.org/10.1093/neuonc/noz126.194.

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Abstract Background: The predictors of a favorable prognosis regarding radiation exposure in patients with glioblastoma have not been studied enough, therefore, the search for factors determining a tumor response to the combined chemoradiation treatment is an actual task at present. DNA repair protein RAD51 homolog 1 (RAD51) is a crucial protein for homologous recombination and its inhibition has been shown to sensitize glioma stem cells to irradiation. Materials and Methods: G01 primary glioblastoma cell culture was obtained from tumor tissues from patient with long term survival (42 month).An irradiation with the energy of 6 MeV was carried out in 1 fraction 20,40,60,80,100,125,150,200,250 Gy with Linac. mRNA expression of the RAD51 gene, XRCC6, XRCC5 genes, relative amount of RAD51 protein and Ku70, Ku80 proteins, proliferative activity of cell culture was evaluated by RT-PCR, Western blotting, MTT-test, respectively.Results: When G01 cells were irradiated with doses from 5 to 180 Gy there was a uniform linear decrease in the population of proliferating cells compared to the control, but at a dose of 200 Gy a rise to 24% was observed. The mRNA expression of the RAD51 gene increased 2 times after irradiation at a dose of 20 Gy compared with control and then increased, reaching a peak at the level of 100,250 Gy. Minimal expression was observed after irradiation at a dose of 20 and also decreased at 200 Gy. The mRNA expression of the XRCC5 gene increased 2-fold after irradiation at a dose of 20 Gy compared with control and then decreased, remaining at the same level. Minimal expression was observed after irradiation at a dose of 200Gy. The relative amount of Ku80 protein reached a peak at a dose of 100 Gy, followed by a decrease. Maximum expression level of mRNA of the XRCC6 gene was observed after irradiation at a dose of 250Gy. The relative amount of Ku70 protein also decreased at 200 Gy and reached a peak at a dose of 250 Gy. Conclusions: After irradiation transplantable G01 cell culture from patient with long term survival a linear decrease in proliferative activity depending on the dose was observed with an decrease in mRNA expression of Rad51 gene and the relative amount of RAD51,Ku70 in dose level of 200Gy that could potentially indicate sensitivity point to radiation exposure in these cell culture. Further studies using cell cultures from patients may help in understanding the mechanisms of radioresistance and radiosensitivity in glioblastoma.The research was supported financially by RFBR (Project No. 18-29-01061).

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Wang, Xin, Ana-Maria Dragoi, Matthew Riolo, Michelle (Xiao) Peng, and Wen-Ming Chu. "A novel role for Ku70 in differential regulation of pro-inflammatory cytokine response to CpG-DNA (89.22)." Journal of Immunology 178, no. 1_Supplement (April 1, 2007): S153. http://dx.doi.org/10.4049/jimmunol.178.supp.89.22.

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Abstract CpG-DNA or synthetic oligodeoxynucleotides containing CpG motif (CpG-ODNs) strongly activate dendritic cells (DCs) and macrophages to produce proinflammatory cytokines including IL-6, IL-12 and TNF?. It has been suggested that CpG-DNA activates TLR9, which in turn recruits MyD88, IRAK4, IRAK1 and TRAF6, leading to activation of AP-1 and NF-?B, which are critical for immune gene expression. However, whether DNA binding proteins are involved in activation of the CpG-DNA pathway remains elusive. Here we demonstrate that Ku70 is involved in this process. Administration of CpG-ODN into Ku70-deficient mice and in vitro stimulation of bone marrow-derived DCs (BMDCs) as well as bone marrow-derived macrophages (BMDMs) from these mice resulted in defective induction of IL-6 and TNF?. Loss of Ku70 impaired activation of its downstream kinases, the I?B kinase (IKK) and the mitogen-activated protein kinases (MAPKs), by CpG-ODN. Intriguingly, in macrophages ablation of Ku70 largely abrogated IRF1 expression and subsequent IL-12 response to CpG-ODN; in BMDCs Ku70 deficiency only reduced IL-12 response to CpG-ODN at a low dose within early treatments, and had no apparent effects on this response in late treatments. Thus, our results suggest that Ku70 is important for differential regulation of inflammatory cytokine response to CpG-DNA. In addition, we have identified a mechanism for involvement of Ku70 in activation of the CpG-DNA pathway.

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Jia, Qiang, Yanhe Li, Desheng Xu, Zhenjiang Li, Zhiyuan Zhang, Yipei Zhang, Dong Liu, Xiaomin Liu, Peiyu Pu, and Chunsheng Kang. "Radiosensitivity of glioma to Gamma Knife treatment enhanced in vitro and in vivo by RNA interfering Ku70 that is mediated by a recombinant adenovirus." Journal of Neurosurgery 113, Special_Supplement (December 2010): 228–35. http://dx.doi.org/10.3171/2010.7.gks10972.

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Object The authors sought to evaluate modification of the radiation response of C6 glioma cells in vitro and in vivo by inhibiting the expression of Ku70. To do so they investigated the effect of gene transfer involving a recombinant replication-defective adenovirus containing Ku70 short hairpin RNA (Ad-Ku70shRNA) combined with Gamma Knife treatment (GKT). Methods First, Ad-Ku70shRNA was transfected into C6 glioma cells and the expression of Ku70 was measured using Western blot analysis. In vitro, phenotypical changes in C6 cells, including proliferation, cell cycle modification, invasion ability, and apoptosis were evaluated using the MTT (3′(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, Western blot analysis, and cell flow cytometry. In vivo, parental C6 cells transfected with Ad-Ku70shRNA were implanted stereotactically into the right caudate nucleus in Sprague-Dawley rats. After GKS, apoptosis was analyzed using the TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling) method. The inhibitory effects on growth and invasion that were induced by expression of proliferating cell nuclear antigen and matrix metalloproteinase–9 were determined using immunohistochemical analyses. Results The expression of Ku70 was clearly inhibited in C6 cells after transfection with Ad-Ku70shRNA. In vitro following transfection, the C6 cells showed improved responses to GKT, including suppression of proliferation and invasion as well as an increased apoptosis index. In vivo following transfection of Ad-Ku70shRNA, the therapeutic efficacy of GKT in rats with C6 gliomas was greatly enhanced and survival times in these animals were prolonged. Conclusions Our data support the potential for downregulation of Ku70 expression in enhancing the radiosensitivity of gliomas. The findings of our study indicate that targeted gene therapy–mediated inactivation of Ku70 may represent a promising strategy in improving the radioresponsiveness of gliomas to GKT.

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Manis, John P., Yansong Gu, Rusty Lansford, Eiichiro Sonoda, Roger Ferrini, Laurie Davidson, Klaus Rajewsky, and Frederick W. Alt. "Ku70 Is Required for Late B Cell Development and Immunoglobulin Heavy Chain Class Switching." Journal of Experimental Medicine 187, no. 12 (June 15, 1998): 2081–89. http://dx.doi.org/10.1084/jem.187.12.2081.

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Immunoglobulin (Ig) heavy chain (HC) class switch recombination (CSR) is a late B cell process that involves intrachromosomal DNA rearrangement. Ku70 and Ku80 form a DNA end-binding complex required for DNA double strand break repair and V(D)J recombination. Ku70−/− (K70T) mice, like recombination activating gene (RAG)-1– or RAG-2–deficient (R1T or R2T) mice, have impaired B and T cell development at an early progenitor stage, which is thought to result at least in part from defective V(D)J recombination (Gu, Y., K.J. Seidl, G.A. Rathbun, C. Zhu, J.P. Manis, N. van der Stoep, L. Davidson, H.L. Cheng, J.M. Sekiguchi, K. Frank, et al. 1997. Immunity. 7:653–665; Ouyang, H., A. Nussenzweig, A. Kurimasa, V.C. Soares, X. Li, C. Cordon-Cardo, W. Li, N. Cheong, M. Nussenzweig, G. Iliakis, et al. 1997. J. Exp. Med. 186:921–929). Therefore, to examine the potential role of Ku70 in CSR, we generated K70T mice that carry a germline Ig HC locus in which the JH region was replaced with a functionally rearranged VH(D)JH and Ig λ light chain transgene (referred to as K70T/HL mice). Previously, we have shown that B cells from R1T or R2T mice carrying these rearranged Ig genes (R1T/HL or R2T/HL mice) can undergo CSR to IgG isotypes (Lansford, R., J. Manis, E. Sonoda, K. Rajewsky, and F. Alt. 1998. Int. Immunol. 10:325–332). K70T/HL mice had significant numbers of peripheral surface IgM+ B cells, which generated serum IgM levels similar to those of R2T/HL mice. However, in contrast to R2T/HL mice, K70T/HL mice had no detectable serum IgG isotypes. In vitro culture of K70T/HL B cells with agents that induce CSR in normal or R2T/HL B cells did lead to the induction of germline CH transcripts, indicating that initial signaling pathways for CSR were intact in K70T/HL cells. However, treatment with such agents did not lead to detectable CSR by K70T/HL B cells, and instead, led to cell death within 72 h. We conclude that Ku70 is required for the generation of B cells that have undergone Ig HC class switching. Potential roles for Ku70 in the CSR process are discussed.

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Gong, Ping, Yuetong Wang, and Yongkui Jing. "Apoptosis Induction byHistone Deacetylase Inhibitors in Cancer Cells: Role of Ku70." International Journal of Molecular Sciences 20, no. 7 (March 30, 2019): 1601. http://dx.doi.org/10.3390/ijms20071601.

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Histone deacetylases (HDACs) are a group of enzymes that regulate gene transcription by controlling deacetylation of histones and non-histone proteins. Overexpression of HDACs is found in some types of tumors and predicts poor prognosis. Five HDAC inhibitors are approved for the treatment of cutaneous T-cell lymphoma, peripheral T-cell lymphoma, and multiple myeloma. Treatment with HDAC inhibitors regulates gene expression with increased acetylated histones with unconfirmed connection with therapy. Apoptosis is a key mechanism by which HDAC inhibitors selectively kill cancer cells, probably due to acetylation of non-histone proteins. Ku70 is a protein that repairs DNA breaks and stabilizes anti-apoptotic protein c-FLIP and proapoptotic protein Bax, which is regulated by acetylation. HDAC inhibitors induce Ku70 acetylation with repressed c-FLIP and activated Bax in cancer cells. Current studies indicate that Ku70 is a potential target of HDAC inhibitors and plays an important role during the induction of apoptosis.

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Singleton, B. K., A. Priestley, H. Steingrimsdottir, D. Gell, T. Blunt, S. P. Jackson, A. R. Lehmann, and P. A. Jeggo. "Molecular and biochemical characterization of xrs mutants defective in Ku80." Molecular and Cellular Biology 17, no. 3 (March 1997): 1264–73. http://dx.doi.org/10.1128/mcb.17.3.1264.

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The gene product defective in radiosensitive CHO mutants belonging to ionizing radiation complementation group 5, which includes the extensively studied xrs mutants, has recently been identified as Ku80, a subunit of the Ku protein and a component of DNA-dependent protein kinase (DNA-PK). Several group 5 mutants, including xrs-5 and -6, lack double-stranded DNA end-binding and DNA-PK activities. In this study, we examined additional xrs mutants at the molecular and biochemical levels. All mutants examined have low or undetectable levels of Ku70 and Ku80 protein, end-binding, and DNA-PK activities. Only one mutant, xrs-6, has Ku80 transcript levels detectable by Northern hybridization, but Ku80 mRNA was detectable by reverse transcription-PCR in most other mutants. Two mutants, xrs-4 and -6, have altered Ku80 transcripts resulting from mutational changes in the genomic Ku80 sequence affecting RNA splicing, indicating that the defects in these mutants lie in the Ku80 gene rather than a gene controlling its expression. Neither of these two mutants has detectable wild-type Ku80 transcript. Since the mutation in both xrs-4 and xrs-6 cells results in severely truncated Ku80 protein, both are likely candidates to be null mutants. Azacytidine-induced revertants of xrs-4 and -6 carried both wild-type and mutant transcripts. The results with these revertants strongly support our model proposed earlier, that CHO-K1 cells carry a copy of the Ku80 gene (XRCC5) silenced by hypermethylation. Site-directed mutagenesis studies indicate that previously proposed ATP-binding and phosphorylation sites are not required for Ku80 activity, whereas N-terminal deletions of more than the first seven amino acids result in severe loss of activities.

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Takiguchi, Yuichi, Akihiro Kurimasa, Fanqing Chen, Paige E. Pardington, Takayuki Kyama, Richard T. Okinaka, Robert Moyzis, and David J. Chen. "Genomic Structure and Chromosomal Assignment of the Mouse Ku70 Gene." Genomics 35, no. 1 (July 1996): 129–35. http://dx.doi.org/10.1006/geno.1996.0331.

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21

Chang, Wen-Chi, Yung-Kai Wang, Pei-Feng Liu, Yu-Fang Tsai, Lih-Ren Kong, Chi-Kai Lin, Chang-Hsien Yang, and Rong-Long Pan. "Regulation of Ku gene promoters in Arabidopsis by hormones and stress." Functional Plant Biology 35, no. 4 (2008): 265. http://dx.doi.org/10.1071/fp07249.

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The Ku70/Ku80 heterodimer plays a crucial role in non-homologous end-joining during DNA repair, and is also involved in multiple cellular processes such as telomere maintenance, transcription, and apoptosis. In this study, we investigate the regulation of AtKu genes in higher plants. Promoters of the AtKu70 and AtKu80 were isolated from Arabidopsis and their activities characterised using GUS reporter constructs. AtKu promoter activities were relatively higher in hypocotyls and cotyledons upon germination and in stigma and siliques as well at their early developing stages. Furthermore, AtKu promoter activities could be enhanced by gibberellic acid, auxins, and jasmonic acid, but repressed by abscisic acid, salicylic acid, heat, drought and cold, respectively. Deletion analysis demonstrates minimal lengths of ~400 bp and 600 bp upstream of transcription start site for functional promoters of AtKu70 and AtKu80, respectively. Taken together, expressions of Ku genes are regulated both by developmental programs as well as by plant hormones and environmental stresses.

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He, Yi, Qingpei Liu, Yanchun Shao, and Fusheng Chen. "Erratum to: ku70 and ku80 null mutants improve the gene targeting frequency in Monascus ruber M7." Applied Microbiology and Biotechnology 97, no. 11 (April 27, 2013): 5175–76. http://dx.doi.org/10.1007/s00253-013-4916-8.

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23

Takahashi, Tadashi, Tsutomu Masuda, and Yasuji Koyama. "Enhanced gene targeting frequency in ku70 and ku80 disruption mutants of Aspergillus sojae and Aspergillus oryzae." Molecular Genetics and Genomics 275, no. 5 (February 10, 2006): 460–70. http://dx.doi.org/10.1007/s00438-006-0104-1.

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24

Krappmann, Sven, Christoph Sasse, and Gerhard H. Braus. "Gene Targeting in Aspergillus fumigatus by Homologous Recombination Is Facilitated in a Nonhomologous End- Joining-Deficient Genetic Background." Eukaryotic Cell 5, no. 1 (January 2006): 212–15. http://dx.doi.org/10.1128/ec.5.1.212-215.2006.

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ABSTRACT The akuA gene encoding the Ku70 component of the nonhomologous end-joining machinery was deleted in the opportunistic pathogen Aspergillus fumigatus. No obvious phenotype could be assessed for the corresponding mutant strain but relative frequencies of homologous recombination were increased as deduced from targeting the laccase-encoding abr2 gene.

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25

Zhou, Jenny, and Shu-Ming Li. "Conversion of viridicatic acid to crustosic acid by cytochrome P450 enzyme-catalysed hydroxylation and spontaneous cyclisation." Applied Microbiology and Biotechnology 105, no. 24 (November 11, 2021): 9181–89. http://dx.doi.org/10.1007/s00253-021-11674-4.

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Abstract Cytochrome P450 monooxygenases (P450s) are considered nature’s most versatile catalysts and play a crucial role in regio- and stereoselective oxidation reactions on a broad range of organic molecules. The oxyfunctionalisation of unactivated carbon-hydrogen (C-H) bonds, in particular, represents a key step in the biosynthesis of many natural products as it provides substrates with increased reactivity for tailoring reactions. In this study, we investigated the function of the P450 enzyme TraB in the terrestric acid biosynthetic pathway. We firstly deleted the gene coding for the DNA repair subunit protein Ku70 by using split marker-based deletion plasmids for convenient recycling of the selection marker to improve gene targeting in Penicillium crustosum. Hereby, we reduced ectopic DNA integration and facilitated genetic manipulation in P. crustosum. Afterward, gene deletion in the Δku70 mutant of the native producer P. crustosum and heterologous expression in Aspergillus nidulans with precursor feeding proved the involvement of TraB in the formation of crustosic acid by catalysing the essential hydroxylation reaction of viridicatic acid. Key points •Deletion of Ku70 by using split marker approach for selection marker recycling. •Functional identification of the cytochrome P450 enzyme TraB. •Fulfilling the reaction steps in the terrestric acid biosynthesis.

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Poláková, Ester, Amanda T. S. Albanaz, Alexandra Zakharova, Tatiana S. Novozhilova, Evgeny S. Gerasimov, and Vyacheslav Yurchenko. "Ku80 is involved in telomere maintenance but dispensable for genomic stability in Leishmania mexicana." PLOS Neglected Tropical Diseases 15, no. 12 (December 29, 2021): e0010041. http://dx.doi.org/10.1371/journal.pntd.0010041.

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Background Telomeres are indispensable for genome stability maintenance. They are maintained by the telomere-associated protein complex, which include Ku proteins and a telomerase among others. Here, we investigated a role of Ku80 in Leishmania mexicana. Leishmania is a genus of parasitic protists of the family Trypanosomatidae causing a vector-born disease called leishmaniasis. Methodology/Principal findings We used the previously established CRISPR/Cas9 system to mediate ablation of Ku80- and Ku70-encoding genes in L. mexicana. Complete knock-outs of both genes were confirmed by Southern blotting, whole-genome Illumina sequencing, and RT-qPCR. Resulting telomeric phenotypes were subsequently investigated using Southern blotting detection of terminal restriction fragments. The genome integrity in the Ku80- deficient cells was further investigated by whole-genome sequencing. Our work revealed that telomeres in the ΔKu80 L. mexicana are elongated compared to those of the wild type. This is a surprising finding considering that in another model trypanosomatid, Trypanosoma brucei, they are shortened upon ablation of the same gene. A telomere elongation phenotype has been documented in other species and associated with a presence of telomerase-independent alternative telomere lengthening pathway. Our results also showed that Ku80 appears to be not involved in genome stability maintenance in L. mexicana. Conclusion/Significance Ablation of the Ku proteins in L. mexicana triggers telomere elongation, but does not have an adverse impact on genome integrity.

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27

Feng, Jie, Weijiao Li, Sheau-Fang Hwang, Bruce D. Gossen, and Stephen E. Strelkov. "Enhanced gene replacement frequency in KU70 disruption strain of Stagonospora nodorum." Microbiological Research 167, no. 3 (March 2012): 173–78. http://dx.doi.org/10.1016/j.micres.2011.05.004.

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Ruis, Brian L., Kazi R. Fattah, and Eric A. Hendrickson. "The Catalytic Subunit of DNA-Dependent Protein Kinase Regulates Proliferation, Telomere Length, and Genomic Stability in Human Somatic Cells." Molecular and Cellular Biology 28, no. 20 (August 18, 2008): 6182–95. http://dx.doi.org/10.1128/mcb.00355-08.

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ABSTRACT The DNA-dependent protein kinase (DNA-PK) complex is a serine/threonine protein kinase comprised of a 469-kDa catalytic subunit (DNA-PKcs) and the DNA binding regulatory heterodimeric (Ku70/Ku86) complex Ku. DNA-PK functions in the nonhomologous end-joining pathway for the repair of DNA double-stranded breaks (DSBs) introduced by either exogenous DNA damage or endogenous processes, such as lymphoid V(D)J recombination. Not surprisingly, mutations in Ku70, Ku86, or DNA-PKcs result in animals that are sensitive to agents that cause DSBs and that are also immune deficient. While these phenotypes have been validated in several model systems, an extension of them to humans has been missing due to the lack of patients with mutations in any one of the three DNA-PK subunits. The worldwide lack of patients suggests that during mammalian evolution this complex has become uniquely essential in primates. This hypothesis was substantiated by the demonstration that functional inactivation of either Ku70 or Ku86 in human somatic cell lines is lethal. Here we report on the functional inactivation of DNA-PKcs in human somatic cells. Surprisingly, DNA-PKcs does not appear to be essential, although the cell line lacking this gene has profound proliferation and genomic stability deficits not observed for other mammalian systems.

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Sundaresan, Nagalingam R., Sadhana A. Samant, Vinodkumar B. Pillai, Senthilkumar B. Rajamohan, and Mahesh P. Gupta. "SIRT3 Is a Stress-Responsive Deacetylase in Cardiomyocytes That Protects Cells from Stress-Mediated Cell Death by Deacetylation of Ku70." Molecular and Cellular Biology 28, no. 20 (August 18, 2008): 6384–401. http://dx.doi.org/10.1128/mcb.00426-08.

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ABSTRACT There are seven SIRT isoforms in mammals, with diverse biological functions including gene regulation, metabolism, and apoptosis. Among them, SIRT3 is the only sirtuin whose increased expression has been shown to correlate with an extended life span in humans. In this study, we examined the role of SIRT3 in murine cardiomyocytes. We found that SIRT3 is a stress-responsive deacetylase and that its increased expression protects myocytes from genotoxic and oxidative stress-mediated cell death. We show that, like human SIRT3, mouse SIRT3 is expressed in two forms, a ∼44-kDa long form and a ∼28-kDa short form. Whereas the long form is localized in the mitochondria, nucleus, and cytoplasm, the short form is localized exclusively in the mitochondria of cardiomyocytes. During stress, SIRT3 levels are increased not only in mitochondria but also in the nuclei of cardiomyocytes. We also identified Ku70 as a new target of SIRT3. SIRT3 physically binds to Ku70 and deacetylates it, and this promotes interaction of Ku70 with the proapoptotic protein Bax. Thus, under stress conditions, increased expression of SIRT3 protects cardiomyocytes, in part by hindering the translocation of Bax to mitochondria. These studies underscore an essential role of SIRT3 in the survival of cardiomyocytes in stress situations.

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Masson, Christel, Stéphanie Bury-Moné, Elvire Guiot, Asier Saez-Cirion, Damien Schoëvaërt-Brossault, Corinne Brachet-Ducos, Olivier Delelis, Frédéric Subra, Laurence Jeanson-Leh, and Jean-François Mouscadet. "Ku80 Participates in the Targeting of Retroviral Transgenes to the Chromatin of CHO Cells." Journal of Virology 81, no. 15 (May 16, 2007): 7924–32. http://dx.doi.org/10.1128/jvi.02015-06.

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ABSTRACT The heterodimer Ku70/80 Ku is the DNA-binding component of the DNA-PK complex required for the nonhomologous end-joining pathway. It participates in numerous nuclear processes, including telomere and chromatin structure maintenance, replication, and transcription. Ku interacts with retroviral preintegration complexes and is thought to interfere with the retroviral replication cycle, in particular the formation of 2-long terminal repeat (LTR) viral DNA circles, viral DNA integration, and transcription. We describe here the effect of Ku80 on both provirus integration and the resulting transgene expression in cells transduced with retroviral vectors. We found that transgene expression was systematically higher in Ku80-deficient xrs6 cells than in Ku80-expressing CHO cells. This higher expression was observed irrespective of the presence of the viral LTR and was also not related to the nature of the promoter. Real-time PCR monitoring of the early viral replicative steps demonstrated that the absence of Ku80 does not affect the efficiency of transduction. We analyzed the transgene distributions localization in nucleus by applying a three-dimensional reconstruction model to two-dimensional fluorescence in situ hybridization images. This indicated that the presence of Ku80 resulted in a bias toward the transgenes being located at the periphery of the nucleus associated with their being repressed; in the absence of this factor the transgenes tend to be randomly distributed and actively expressed. Therefore, although not strictly required for retroviral integration, Ku may be involved in targeting retroviral elements to chromatin domains prone to gene silencing.

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Huang, Qingqing, Yang Cao, Zuoyi Liu, Yumei Tan, and Yongxiang Liu. "Efficient gene replacements in ku70 disruption strain of Aspergillus chevalieri var. intermedius." Biotechnology & Biotechnological Equipment 31, no. 1 (November 18, 2016): 16–22. http://dx.doi.org/10.1080/13102818.2016.1251828.

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Bertolini, Luciana R., Marcelo Bertolini, Elizabeth A. Maga, Knut R. Madden, and James D. Murray. "Increased Gene Targeting in Ku70 and Xrcc4 Transiently Deficient Human Somatic Cells." Molecular Biotechnology 41, no. 2 (August 30, 2008): 106–14. http://dx.doi.org/10.1007/s12033-008-9098-8.

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Prall, Wolf C., Akos Czibere, Franck Grall, Luiz F. Zerbini, Markus Jaeger, Dimitrios Spentzos, Towia A. Libermann, Norbert Gattermann, Manuel Aivado, and Rainer Haas. "Age-Related Transcription Levels of KU70 and BIK in CD34+ Hematopoietic Stem and Progenitor Cells." Blood 106, no. 11 (November 16, 2005): 4205. http://dx.doi.org/10.1182/blood.v106.11.4205.4205.

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Abstract There is a higher incidence of hematological clonal stem cell disorders in elderly persons. Age-related alterations of hematopoietic stem and progenitor cells (HSC) may represent one factor underlying this observation. However, the molecular changes related to stem cell aging are largely unknown. Therefore, we scrutinized gene expression patterns of HSC from umbilical cord blood (CB) as well as bone marrow from young (BM-Y, mean age 32,8, SD 12,4) and old (BM-O, mean age 88,8, SD 4,4) healthy donors. CD34+ HSC were isolated via immuno-magnetic separation and CD34+ purity was evaluated by FACS analysis. Thereafter we performed cDNA array analyses on a first set of samples (n=18). We found that the BCL2-interacting killer gene (BIK) and the gene encoding KU Antigen 70kD (KU70) show age-related mRNA expression levels. BIK is a proapoptotic gene and its expression was positively correlated with donor’s age, i.e. lowest in CB, 1.8-fold higher in BM-Y and 4.2-fold higher in BM-O. KU70 is a DNA repair gene and part of the DNA dependent protein kinase (DNA-PK). Its expression was negatively correlated with donor’s age showing highest expression levels in CB, 2.2-fold lower levels in BM-Y and 5.3-fold lower levels in BM-O. These findings were confirmed with a second set of samples (n=16) by means of quantitative RT-PCR. Elucidation of age-dependent molecular alteration in healthy HSC might facilitate a better understanding of hematological malignancies in elderly persons.

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Fattah, F. J., N. F. Lichter, K. R. Fattah, S. Oh, and E. A. Hendrickson. "Ku70, an essential gene, modulates the frequency of rAAV-mediated gene targeting in human somatic cells." Proceedings of the National Academy of Sciences 105, no. 25 (June 18, 2008): 8703–8. http://dx.doi.org/10.1073/pnas.0712060105.

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Ohno, Masae, Jun Komakine, Eiko Suzuki, Makoto Nishizuka, Shigehiro Osada та Masayoshi Imagawa. "Interleukin enhancer-binding factor 3 functions as a liver receptor homologue-1 co-activator in synergy with the nuclear receptor co-activators PRMT1 and PGC-1α". Biochemical Journal 437, № 3 (13 липня 2011): 531–40. http://dx.doi.org/10.1042/bj20101793.

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LRH-1 (liver receptor homologue-1), a transcription factor and member of the nuclear receptor superfamily, regulates the expression of its target genes, which are involved in bile acid and cholesterol homoeostasis. However, the molecular mechanisms of transcriptional control by LRH-1 are not completely understood. Previously, we identified Ku80 and Ku70 as LRH-1-binding proteins and reported that they function as co-repressors. In the present study, we identified an additional LRH-1-binding protein, ILF3 (interleukin enhancer-binding factor 3). ILF3 formed a complex with LRH-1 and the other two nuclear receptor co-activators PRMT1 (protein arginine methyltransferase 1) and PGC-1α (peroxisome proliferator-activated receptor γ co-activator-1α). We demonstrated that ILF3, PRMT1 and PGC-1α were recruited to the promoter region of the LRH-1-regulated SHP (small heterodimer partner) gene, encoding one of the nuclear receptors. ILF3 enhanced SHP gene expression in co-operation with PRMT1 and PGC-1α through the C-terminal region of ILF3. In addition, we found that the small interfering RNA-mediated down-regulation of ILF3 expression led to a reduction in the occupancy of PGC-1α at the SHP promoter and SHP expression. Taken together, our results suggest that ILF3 functions as a novel LRH-1 co-activator by acting synergistically with PRMT1 and PGC-1α, thereby promoting LRH-1-dependent gene expression.

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Bystricky, Kerstin, Haico Van Attikum, Maria-Dolores Montiel, Vincent Dion, Lutz Gehlen, and Susan M. Gasser. "Regulation of Nuclear Positioning and Dynamics of the Silent Mating Type Loci by the Yeast Ku70/Ku80 Complex." Molecular and Cellular Biology 29, no. 3 (December 1, 2008): 835–48. http://dx.doi.org/10.1128/mcb.01009-08.

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ABSTRACT We have examined the hypothesis that the highly selective recombination of an active mating type locus (MAT) with either HMLα or HMR a is facilitated by the spatial positioning of relevant sequences within the budding yeast (Saccharomyces cerevisiae) nucleus. However, both position relative to the nuclear envelope (NE) and the subnuclear mobility of fluorescently tagged MAT, HML, or HMR loci are largely identical in haploid a and α cells. Irrespective of mating type, the expressed MAT locus is highly mobile within the nuclear lumen, while silent loci move less and are found preferentially near the NE. The perinuclear positions of HMR and HML are strongly compromised in strains lacking the Silent information regulator, Sir4. However, HMLα, unlike HMR a and most telomeres, shows increased NE association in a strain lacking yeast Ku70 (yKu70). Intriguingly, we find that the yKu complex is associated with HML and HMR sequences in a mating-type-specific manner. Its abundance decreases at the HMLα donor locus and increases transiently at MAT a following DSB induction. Our data suggest that mating-type-specific binding of yKu to HMLα creates a local chromatin structure competent for recombination, which cooperates with the recombination enhancer to direct donor choice for gene conversion of the MAT a locus.

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Kim, Sangkyu, Eric Simon, Leann Myers, L. Lee Hamm, and S. Michal Jazwinski. "Programmed Cell Death Genes Are Linked to Elevated Creatine Kinase Levels in Unhealthy Male Nonagenarians." Gerontology 62, no. 5 (2016): 519–29. http://dx.doi.org/10.1159/000443793.

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Declining health in the oldest-old takes an energy toll for the simple maintenance of body functions. The underlying mechanisms, however, differ in males and females. In females, the declines are explained by loss of muscle mass; but this is not the case in males, in whom they are associated with increased levels of circulating creatine kinase. This relationship raises the possibility that muscle damage rather than muscle loss is the cause of the increased energy demands of unhealthy aging in males. We have now examined factors that contribute to the increase in creatine kinase. Much of it (60%) can be explained by a history of cardiac problems and lower kidney function, while being mitigated by moderate physical activity, reinforcing the notion that tissue damage is a likely source. In a search for genetic risk factors associated with elevated creatine kinase, the Ku70 gene XRCC6 and the ceramide synthase gene LASS1 were investigated because of their roles in telomere length and longevity and healthy aging, respectively. Single nucleotide polymorphisms in these two genes were independently associated with creatine kinase levels. The XRCC6 variant was epistatic to one of the LASS1 variants but not to the other. These gene variants have potential regulatory activity. Ku70 is an inhibitor of the proapoptotic Bax, while the product of Lass1, ceramide, operates in both caspase-dependent and -independent pathways of programmed cell death, providing a potential cellular mechanism for the effects of these genes on tissue damage and circulating creatine kinase.

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Kusano, K. "Sterility of Drosophila with Mutations in the Bloom Syndrome Gene--Complementation by Ku70." Science 291, no. 5513 (March 30, 2001): 2600–2602. http://dx.doi.org/10.1126/science.291.5513.2600.

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39

He, F., R. Shao, M. Urano, S. Leibel, P. H. Gutin, and G. C. Li. "Adenovirus-mediated antisense KU70 gene transfer increases the radiosensitivity of human malignant gliomas." International Journal of Radiation Oncology*Biology*Physics 54, no. 2 (October 2002): 178. http://dx.doi.org/10.1016/s0360-3016(02)03366-7.

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40

Basu, Sanjay, Azadeh Aryan, Justin M. Overcash, Glady Hazitha Samuel, Michelle A. E. Anderson, Timothy J. Dahlem, Kevin M. Myles, and Zach N. Adelman. "Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis in Aedes aegypti." Proceedings of the National Academy of Sciences 112, no. 13 (March 16, 2015): 4038–43. http://dx.doi.org/10.1073/pnas.1502370112.

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Conventional control strategies for mosquito-borne pathogens such as malaria and dengue are now being complemented by the development of transgenic mosquito strains reprogrammed to generate beneficial phenotypes such as conditional sterility or pathogen resistance. The widespread success of site-specific nucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in model organisms also suggests that reprogrammable gene drive systems based on these nucleases may be capable of spreading such beneficial phenotypes in wild mosquito populations. Using the mosquito Aedes aegypti, we determined that mutations in the FokI domain used in TALENs to generate obligate heterodimeric complexes substantially and significantly reduce gene editing rates. We found that CRISPR/Cas9-based editing in the mosquito Ae. aegypti is also highly variable, with the majority of guide RNAs unable to generate detectable editing. By first evaluating candidate guide RNAs using a transient embryo assay, we were able to rapidly identify highly effective guide RNAs; focusing germ line-based experiments only on this cohort resulted in consistently high editing rates of 24–90%. Microinjection of double-stranded RNAs targeting ku70 or lig4, both essential components of the end-joining response, increased recombination-based repair in early embryos as determined by plasmid-based reporters. RNAi-based suppression of Ku70 concurrent with embryonic microinjection of site-specific nucleases yielded consistent gene insertion frequencies of 2–3%, similar to traditional transposon- or ΦC31-based integration methods but without the requirement for an initial docking step. These studies should greatly accelerate investigations into mosquito biology, streamline development of transgenic strains for field releases, and simplify the evaluation of novel Cas9-based gene drive systems.

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41

Tavares, K. C. S., C. R. Lazzarotto, S. G. Neto, L. T. Martins, L. H. Aguiar, C. E. M. Calderón, L. P. R. Teixeira, et al. "241 CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR)/Cas9 ASSOCIATED WITH TRANSIENT DEPLETION OF NON-HOMOLOGOUS END-JOINING PATHWAY INCREASED GENE-TARGETING EFFICIENCY IN GOAT FIBROBLASTS." Reproduction, Fertility and Development 28, no. 2 (2016): 252. http://dx.doi.org/10.1071/rdv28n2ab241.

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The transgenic animal platform for the expression of recombinant proteins in the milk offers particularly attractive possibilities. The recent application of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to promote precise genome modifications and DNA editing also allows the targeting of specific DNA sequences in embryos or cells in culture. In addition, the transient knockdown of the NHEJ pathway by RNAi has been shown to increase gene‐targeting (GT) rate in cultured cells (Bertolini et al. 2009 Mol. Biotechnol. 41, 106–114). The CRISPR/Cas9 system was used to target a 15-kb transgene construct into the ROSA26 locus in goat fetal fibroblast cells subjected to a transient RNAi‐induced depletion of the NHEJ Ku70 protein. A polycistronic expression vector was constructed by ligating the coding sequences of 3 antigenic proteins against Brucella abortus linked by self-processing 2A peptides under the regulation of the bovine α-lactalbumin promoter. The final vector also contained the neomycin resistance gene and left and right 2-kb arms homologous to the goat ROSA26 locus. A total of 2 × 105 fibroblast cells at passage 3 from a 50-day fetus were transfected using the Neon Transfection System (Invitrogen/ThermoFisher Scientific, Waltham, MA, USA), according to the following groups: mock control (M); vector-only (V); vector + RNAi against Ku70 (VR); vector + ROSA26‐CRISPR/Cas9 (VC); and vector + RNAi against Ku70 + ROSA26-CRISPR/Cas9 (VCR). After antibiotic selection, colonies were characterised for zygosity, transgene copy number, and off-targets. Mortality rates following cell transfection were 68, 78, 75, 83, and 90%, and the number of colonies after selection was 0, 13, 22, 5, and 8 for the M, V, VR, VC, and VCR groups, respectively. Gene targeting was detected only when the ROSA26‐CRISPR/Cas9 was combined to the vector (VC group, 1 in 22 colonies) or to the vector and RNAi against Ku70 (VCR group, 1 in 8 colonies), with a 2.8-fold increase in GT rate when associating the 3 components (VCR group). No CRISPR/Cas9 off-targets were detected in 7 different sequenced hot spots. One colony from the VC group, harboring a biallelic transgene knock-in, was chosen for use in goat cloning by SCNT following our established procedures (Martins et al. 2015 Reprod. Fertil. Dev. 27, 111). Four viable pregnancies (33.3%) were established, based on the ultrasonographic visualisation of the embryo and heartbeat on Day 26, after the transfer of 144 embryos to 12 female recipient does, demonstrating the developmental potential of the transgenic knock-in donor cells. However, pregnancies were lost up to Day 55 of pregnancy. Our preliminary data suggest that the combined cell transfection of gene target-specific CRISPR/Cas9 and RNAi to knockdown the NHEJ pathway is a viable and efficient approach to produce precise genetically modified goat donor cells carrying mono- and biallelic knock-ins of large size transgene constructs for use in cloning by SCNT. Cloning procedures are underway using biallelic knock-in somatic cells to obtain live offspring, which will be the first step to produce and test a recombinant subunit vaccine against B. abortus.

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42

Yang, Chih-Cheng, Alicia Chung, Chia-Yu Ku, Laurence M. Brill, Roy Williams, and Dieter A. Wolf. "Systems analysis of the prostate tumor suppressor NKX3.1 supports roles in DNA repair and luminal cell differentiation." F1000Research 3 (May 21, 2014): 115. http://dx.doi.org/10.12688/f1000research.3818.1.

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NKX3.1 is a homeobox transcription factor whose function as a prostate tumor suppressor remains insufficiently understood because neither the transcriptional program governed by NKX3.1, nor its interacting proteins have been fully revealed. Using affinity purification and mass spectrometry, we have established an extensive NKX3.1 interactome which contains the DNA repair proteins Ku70, Ku80, and PARP, thus providing a molecular underpinning to previous reports implicating NKX3.1 in DNA repair. Transcriptomic profiling of NKX3.1-negative prostate epithelial cells acutely expressing NKX3.1 revealed a rapid and complex response that is a near mirror image of the gene expression signature of human prostatic intraepithelial neoplasia (PIN). Pathway and network analyses suggested that NKX3.1 actuates a cellular reprogramming toward luminal cell differentiation characterized by suppression of pro-oncogenic c-MYC and interferon-STAT signaling and activation of tumor suppressor pathways. Consistently, ectopic expression of NKX3.1 conferred a growth arrest depending on TNFα and JNK signaling. We propose that the tumor suppressor function of NKX3.1 entails a transcriptional program that maintains the differentiation state of secretory luminal cells and that disruption of NKX3.1 contributes to prostate tumorigenesis by permitting luminal cell de-differentiation potentially augmented by defects in DNA repair.

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43

Li, Xuanchen, Dilaware Khan, Majeed Rana, Daniel Hänggi, and Sajjad Muhammad. "Doxycycline Attenuated Ethanol-Induced Inflammaging in Endothelial Cells: Implications in Alcohol-Mediated Vascular Diseases." Antioxidants 11, no. 12 (December 7, 2022): 2413. http://dx.doi.org/10.3390/antiox11122413.

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Excess alcohol consumption is a potential risk factor for cardiovascular diseases and is linked to accelerated aging. Drug discovery to reduce toxic cellular events of alcohol is required. Here, we investigated the effects of ethanol on human umbilical vein endothelial cells (HUVECs) and explored if doxycycline attenuates ethanol-mediated molecular events in endothelial cells. Initially, a drug screening using a panel of 170 drugs was performed, and doxycycline was selected for further experiments. HUVECs were treated with different concentrations (300 mM and 400 mM) of ethanol with or without doxycycline (10 µg/mL). Telomere length was quantified as telomere to single-copy gene (T/S) ratio. Telomere length and the mRNA expression were quantified by qRT-PCR, and protein level was analyzed by Western blot (WB). Ethanol treatment accelerated cellular aging, and doxycycline treatment recovered telomere length. Pathway analysis showed that doxycycline inhibited mTOR and NFκ-B activation. Doxycycline restored the expression of aging-associated proteins, including lamin b1 and DNA repair proteins KU70 and KU80. Doxycycline reduced senescence and senescence-associated secretory phenotype (SASP) in ethanol-treated HUVECs. In conclusion, we report that ethanol-induced inflammation and aging in HUVECs were ameliorated by doxycycline.

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44

Errami, A., V. Smider, W. K. Rathmell, D. M. He, E. A. Hendrickson, M. Z. Zdzienicka, and G. Chu. "Ku86 defines the genetic defect and restores X-ray resistance and V(D)J recombination to complementation group 5 hamster cell mutants." Molecular and Cellular Biology 16, no. 4 (April 1996): 1519–26. http://dx.doi.org/10.1128/mcb.16.4.1519.

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X-ray-sensitive hamster cells in complementation groups 4, 5, 6, and 7 are impaired for both double-strand break repair and V(D)J recombination. Here we show that in two mutant cell lines (XR-V15B and XR-V9B) from group 5, the genetic defects are in the gene encoding the 86-kDa subunit of the Ku autoantigen, a nuclear protein that binds to the double-stranded DNA ends. These mutants express Ku86 mRNA containing deletions of 138 and 252 bp, respectively, and the encoded proteins contain internal, in-frame deletions of 46 and 84 amino acids. Two X-ray-resistant revertants of XR-V15B expressed two Ku86 transcripts, one with and one without the deletion, suggesting that reversion occurred by activation of a silent wild-type allele. Transfection of full-length cDNA encoding hamster Ku86 into XR-V15B cells resulted in a complete rescue of DNA-end-binding (DEB) activity and Ku70 levels, suggesting that Ku86 stabilizes the Ku70 polypeptide. In addition, cells expressing wild-type levels of DEB activity were fully rescued for X-ray resistance and V(D)J recombination, whereas cells expressing lower levels of DEB activity were only partially rescued. Thus, Ku is an essential component of the pathway(s) utilized for the resolution of DNA double-strand breaks induced by either X rays or V(D)J recombination, and mutations in the Ku86 gene are responsible for the phenotype of group 5 cells.

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45

Bau, Da-Tian, Hsien-Chang Tseng, Chung-Hsing Wang, Chang-Fang Chiu, Chun-Hung Hua, Cheng-Nan Wu, Shiu-Yun Liang, Cheng-Li Wang, Chia-Wen Tsai, and Ming-Hsui Tsai. "Oral cancer and genetic polymorphism of DNA double strand break gene Ku70 in Taiwan." Oral Oncology 44, no. 11 (November 2008): 1047–51. http://dx.doi.org/10.1016/j.oraloncology.2008.02.008.

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46

Qi, Xiliang, Xiaofeng Su, Huiming Guo, Juncang Qi, and Hongmei Cheng. "A ku70 null mutant improves gene targeting frequency in the fungal pathogen Verticillium dahliae." World Journal of Microbiology and Biotechnology 31, no. 12 (October 16, 2015): 1889–97. http://dx.doi.org/10.1007/s11274-015-1907-1.

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47

Takahashi, Tadashi, Feng Jie Jin, Misao Sunagawa, Masayuki Machida, and Yasuji Koyama. "Generation of Large Chromosomal Deletions in Koji Molds Aspergillus oryzae and Aspergillus sojae via a Loop-Out Recombination." Applied and Environmental Microbiology 74, no. 24 (October 24, 2008): 7684–93. http://dx.doi.org/10.1128/aem.00692-08.

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ABSTRACT We established a technique for efficiently generating large chromosomal deletions in the koji molds Aspergillus oryzae and A. sojae by using a ku70-deficient strain and a bidirectional marker. The approach allowed deletion of 200-kb and 100-kb sections of A. oryzae and A. sojae, respectively. The deleted regions contained putative aflatoxin biosynthetic gene clusters. The large genomic deletions generated by a loop-out deletion method (resolution-type recombination) enabled us to construct multiple deletions in the koji molds by marker recycling. No additional sequence remained in the resultant deletion strains, a feature of considerable value for breeding of food-grade microorganisms. Frequencies of chromosomal deletions tended to decrease in proportion to the length of the deletion range. Deletion efficiency was also affected by the location of the deleted region. Further, comparative genome hybridization analysis showed that no unintended deletion or chromosomal rearrangement occurred in the deletion strain. Strains with large deletions that were previously extremely laborious to construct in the wild-type ku70 + strain due to the low frequency of homologous recombination were efficiently obtained from Δku70 strains in this study. The technique described here may be broadly applicable for the genomic engineering and molecular breeding of filamentous fungi.

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48

Cheng, Yu-Wen, Chien-Ju Lin, Shih-Hsun Kuo, Ann-Shung Lieu, Chee-Yin Chai, Hung-Pei Tsai, and Aij-Lie Kwan. "miR-3059-3p Regulates Glioblastoma Multiforme Radiosensitivity Enhancement through the Homologous Recombination Pathway of DNA Repair." Journal of Oncology 2022 (September 21, 2022): 1–10. http://dx.doi.org/10.1155/2022/7250278.

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Background. Glioblastoma multiforme (GBM) is one of the most deadly and recalcitrant illnesses of the neurocentral nervous system in humans. MicroRNAs (miRNAs) are a class of noncoding RNAs that play important roles in the regulation of gene expression and biological processes, including radiosensitivity. In this study, we demonstrated the relationship between miR-3059-3p and radiation in GBM. Materials and Methods. Radioresistant (RR) cells were obtained by exposing GBM8401 cells to 80 Gy radiation in 20 weekly 4 Gy fractions. miR-3059-3p mRNA and DNA replication helicase/nuclease 2 (DNA2) protein expressions were detected using real-time polymerase chain reaction and immunoblotting. Using flow cytometry, colony formation and apoptosis were identified using miR-3059-3p mimic, miR-3059-3p inhibitor, DNA2 siRNA, and DNA2 plasmid. Immunoblotting was used to detect DNA repair proteins. Results. Low levels of miR-3059-3p and high levels of DNA2 were observed in RR cells. Colony formation and apoptosis assays revealed that miR-3059-3p targeted DNA2 to regulate radioresistance. Immunoblotting revealed that miR-3059-3p regulated the homologous recombination (HR) pathway (Rad51 and Rad52) but not the nonhomologous end joining pathway (ku70 and ku80). Conclusion. Downregulation of DNA2 via miR-3059-3p enhanced the radiosensitivity of GBM cells through the inhibition of the HR pathway.

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49

Gago-Fuentes, Raquel, and Valentyn Oksenych. "Non-Homologous End Joining Factors XLF, PAXX and DNA-PKcs Maintain the Neural Stem and Progenitor Cell Population." Biomolecules 11, no. 1 (December 28, 2020): 20. http://dx.doi.org/10.3390/biom11010020.

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Non-homologous end-joining (NHEJ) is a major DNA repair pathway in mammalian cells that recognizes, processes and fixes DNA damage throughout the cell cycle and is specifically important for homeostasis of post-mitotic neurons and developing lymphocytes. Neuronal apoptosis increases in the mice lacking NHEJ factors Ku70 and Ku80. Inactivation of other NHEJ genes, either Xrcc4 or Lig4, leads to massive neuronal apoptosis in the central nervous system (CNS) that correlates with embryonic lethality in mice. Inactivation of either Paxx, Mri or Dna-pkcs NHEJ gene results in normal CNS development due to compensatory effects of Xlf. Combined inactivation of Xlf/Paxx, Xlf/Mri and Xlf/Dna-pkcs, however, results in late embryonic lethality and high levels of apoptosis in CNS. To determine the impact of NHEJ factors on the early stages of neurodevelopment, we isolated neural stem and progenitor cells from mouse embryos and investigated proliferation, self-renewal and differentiation capacity of these cells lacking either Xlf, Paxx, Dna-pkcs, Xlf/Paxx or Xlf/Dna-pkcs. We found that XRCC4-like factor (XLF), DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and paralogue of XRCC4 and XLF (PAXX) maintain the neural stem and progenitor cell populations and neurodevelopment in mammals, which is particularly evident in the double knockout models.

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50

Brodsky, Michael H., Brian T. Weinert, Garson Tsang, Yikang S. Rong, Nadine M. McGinnis, Kent G. Golic, Donald C. Rio, and Gerald M. Rubin. "Drosophila melanogaster MNK/Chk2 and p53 Regulate Multiple DNA Repair and Apoptotic Pathways following DNA Damage." Molecular and Cellular Biology 24, no. 3 (February 1, 2004): 1219–31. http://dx.doi.org/10.1128/mcb.24.3.1219-1231.2004.

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ABSTRACT We have used genetic and microarray analysis to determine how ionizing radiation (IR) induces p53-dependent transcription and apoptosis in Drosophila melanogaster. IR induces MNK/Chk2-dependent phosphorylation of p53 without changing p53 protein levels, indicating that p53 activity can be regulated without an Mdm2-like activity. In a genome-wide analysis of IR-induced transcription in wild-type and mutant embryos, all IR-induced increases in transcript levels required both p53 and the Drosophila Chk2 homolog MNK. Proapoptotic targets of p53 include hid, reaper, sickle, and the tumor necrosis factor family member Eiger. Overexpression of Eiger is sufficient to induce apoptosis, but mutations in Eiger do not block IR-induced apoptosis. Animals heterozygous for deletions that span the reaper, sickle, and hid genes exhibited reduced IR-dependent apoptosis, indicating that this gene complex is haploinsufficient for induction of apoptosis. Among the genes in this region, hid plays a central, dosage-sensitive role in IR-induced apoptosis. p53 and MNK/Chk2 also regulate DNA repair genes, including two components of the nonhomologous end-joining repair pathway, Ku70 and Ku80. Our results indicate that MNK/Chk2-dependent modification of Drosophila p53 activates a global transcriptional response to DNA damage that induces error-prone DNA repair as well as intrinsic and extrinsic apoptosis pathways.

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