Measuring protein stoichiometry with single-molecule imaging in Xenopus egg extracts.

In human cells, DNA double-strand breaks are rapidly bound by the highly abundant non-homologous end joining (NHEJ) factor Ku70/Ku80 (Ku). Cellular imaging and structural data revealed a single Ku molecule is bound to a free DNA end and yet the mechanism regulating Ku remains unclear. Here, we describe how to utilize the cell-free Xenopus laevis egg extract system in conjunction with single-molecule microscopy to investigate regulation of Ku stoichiometry during non-homologous end joining. Egg extract is an excellent model system to study DNA repair as it contains the soluble proteome including core and accessory NHEJ factors, and efficiently repairs double-strand breaks in an NHEJ-dependent manner. To examine the Ku stoichiometry in the extract system, we developed a single-molecule photobleaching assay, which reports on the number of stable associated Ku molecules by monitoring the intensity of fluorescently labeled Ku molecules bound to double-stranded DNA over time. Photobleaching is distinguishable as step decreases in fluorescence intensity and the number of photobleaching events indicate fluorophore stoichiometry. In this paper we describe sample preparation, experimental methodology, and data analysis to discern Ku stoichiometry and the regulatory mechanism controlling its loading. These approaches can be readily adopted to determine stoichiometry of molecular factors within other macromolecular complexes.

Negative feedback loop in the activation of non-homologous end joining DNA repair pathway in Helicobacter pylori infected patients with gastritis.

This study aimed to investigate the activation of error-prone DNA repair pathway in response to Helicobacter pylori infection. Relative changes in the expression levels of genes involved in the non-homologous end-joining pathway (NHEJ) in H. pylori-infected (Cases) and non-infected patients (Controls) with chronic gastritis were measured. A significant increase in the relative expression level of TP53, and significant decrease in the relative transcription of lncRNA LINP1 and XRCC5 were detected in the case group. The transcription of Lig4 and XRCC6 was increased in the case group, which was not statistically significant. The Spearman's Correlation Coefficient analysis showed a significant positive-correlation between the transcriptional levels of LINP1 and XRCC4/XRCC5/Lig4, and between XRCC5 and TP53/Lig4 both in the case and control groups. Moreover, a significant positive correlation between LinP1 and XRCC6 in the case, and a significant positive correlation between XRCC4 and Lig4, and a negative correlation between TP53 and LinP1/XRCC4/XRCC5 in the control group was detected. Although a relative difference was detected in transcriptional levels of the NHEJ gene mediators, downregulation of LinP1 in H. pylori-infected patients proposed the activation of a negative feedback loop, which may interfere with the NHEJ activity at the early stages of gastritis.

Olaparib combined with CDK12-IN-3 to promote genomic instability and cell death in ovarian cancer.

Large-scale phase III clinical trials of Olaparib have revealed benefits for ovarian cancer patients with BRCA gene mutations or homologous recombination deficiency (HRD). However, fewer than 50% of ovarian cancer patients have both BRCA mutations and HRD. Therefore, improving the effect of Olaparib in HR-proficient patients is of great clinical value. Here, a combination strategy comprising Olaparib and CDK12-IN-3 effectively inhibited the growth of HR-proficient ovarian cancer in cell line, patient-derived organoid (PDO), and mouse xenograft models. Furthermore, the combination strategy induced severe DNA double-strand break (DSB) formation, increased NHEJ activity in the G2 phase, and reduced HR activity in cancer cells. Mechanistically, the combination treatment impaired Ku80 poly(ADP-ribosyl)ation (PARylation) and phosphorylation, resulting in PARP1-Ku80 complex dissociation. After dissociation, Ku80 occupancy at DSBs and the resulting Ku80-primed NHEJ activity were increased. Owing to Ku80-mediated DNA end protection, MRE11 and Rad51 foci formation was inhibited after the combination treatment, suggesting that this treatment suppressed HR activity. Intriguingly, the combination strategy expedited cGAS nuclear relocalization, further suppressing HR and, conversely, increasing genomic instability. Moreover, the inhibitory effect on cell survival persisted after drug withdrawal. These findings provide a rationale for the clinical application of CDK12-IN-3 in combination with Olaparib.

EX527, a sirtuins 1 inhibitor, sensitizes T-cell leukemia to death receptor-mediated apoptosis by downregulating cellular FLICE inhibitory protein.

Death receptor-mediated extrinsic apoptosis system had been developed as a promising therapeutic strategy in clinical oncology, such as TRAIL therapy. However, multiple studies have demonstrated that TRAIL resistance is the biggest problem for disappointing clinical trials despite preclinical success. Targeting cellular FLICE inhibitory protein (cFLIP) is one strategy of combinatorial therapies to overcome resistance to DR-mediated apoptosis due to its negative regulator of extrinsic apoptosis. E × 527 (Selisistat) is a specific inhibitor of SIRT1 activity with safe and well tolerance in clinical trials. Here, we show that E × 527 could strengthen significantly activation of rhFasL-mediated apoptotic signaling pathway and increased apoptotic rate of T leukemia cells with high expression of cFLIP. Mechanically, Inhibition of SIRT1 by E × 527 increased polyubiquitination level of cFLIP via increasing acetylation of Ku70, which could promote proteosomal degradation of cFLIP protein. It implied that combinatorial therapies of E × 527 plus TRAIL may have a potential as a novel clinical application for TRAIL-resistant hematologic malignancies.

ER-associated degradation ligase HRD1 links ER stress to DNA damage repair by modulating the activity of DNA-PKcs.

Proteostasis and genomic integrity are respectively regulated by the endoplasmic reticulum-associated protein degradation (ERAD) and DNA damage repair signaling pathways, with both pathways essential for carcinogenesis and drug resistance. How these signaling pathways coordinate with each other remains unexplored. We found that ER stress specifically induces the DNA-PKcs-regulated nonhomologous end joining (NHEJ) pathway to amend DNA damage and impede cell death. Intriguingly, sustained ER stress rapidly decreased the activity of DNA-PKcs and DNA damage accumulated, facilitating a switch from adaptation to cell death. This DNA-PKcs inactivation was caused by increased KU70/KU80 protein degradation. Unexpectedly, the ERAD ligase HRD1 was found to efficiently destabilize the classic nuclear protein HDAC1 in the cytoplasm, by catalyzing HDAC1's polyubiquitination at lysine 74, at a late stage of ER stress. By abolishing HDAC1-mediated KU70/KU80 deacetylation, HRD1 transmits ER signals to the nucleus. The resulting enhanced KU70/KU80 acetylation provides binding sites for the nuclear E3 ligase TRIM25, resulting in the promotion of polyubiquitination and the degradation of KU70/KU80 proteins. Both in vitro and in vivo cancer models showed that genetic or pharmacological inhibition of HADC1 or DNA-PKcs sensitizes colon cancer cells to ER stress inducers, including the Food and Drug Administration-approved drug celecoxib. The antitumor effects of the combined approach were also observed in patient-derived xenograft models. These findings identify a mechanistic link between ER stress (ERAD) in the cytoplasm and DNA damage (NHEJ) pathways in the nucleus, indicating that combined anticancer strategies may be developed that induce severe ER stress while simultaneously inhibiting KU70/KU80/DNA-PKcs-mediated NHEJ signaling.

PD-L1 deglycosylation promotes its nuclear translocation and accelerates DNA double-strand-break repair in cancer.

Resistance to radiotherapy is a major barrier during cancer treatment. Here using genome-scale CRISPR/Cas9 screening, we identify CD274 gene, which encodes PD-L1, to confer lung cancer cell resistance to ionizing radiation (IR). Depletion of endogenous PD-L1 delays the repair of IR-induced DNA double-strand breaks (DSBs) and PD-L1 loss downregulates non-homologous end joining (NHEJ) while overexpression of PD-L1 upregulates NHEJ. IR induces translocation of PD-L1 from the membrane into nucleus dependent on deglycosylation of PD-L1 at N219 and CMTM6 and leads to PD-L1 recruitment to DSBs foci. PD-L1 interacts with Ku in the nucleus and enhances Ku binding to DSB DNA. The interaction between the IgC domain of PD-L1 and the core domain of Ku is required for PD-L1 to accelerate NHEJ-mediated DSB repair and produce radioresistance. Thus, PD-L1, in addition to its immune inhibitory activity, acts as mechanistic driver for NHEJ-mediated DSB repair in cancer.

Compared to other NHEJ factors, DNA-PK protein and RNA levels are markedly increased in all higher primates, but not in prosimians or other mammals.

The DNA dependent protein kinase (DNA-PK) initiates non-homologous recombination (NHEJ), the predominate DNA double-strand break (DSBR) pathway in higher vertebrates. It has been known for decades that the enzymatic activity of DNA-PK [that requires its three component polypeptides, Ku70, Ku80 (that comprise the DNA-end binding Ku heterodimer), and the catalytic subunit (DNA-PKcs)] is present in humans at 10-50 times the level observed in other mammals. Here, we show that the high level of DNA-PKcs protein expression appears evolutionarily in mammals between prosimians and higher primates. Moreover, the RNAs encoding the three component polypeptides of DNA-PK are present at similarly high levels in hominids, new-, and old-world monkeys, but expression of these RNAs in prosimians is ∼5-50 fold less, analogous to the levels observed in other non-primate species. This is reminiscent of the appearance of Alu repeats in primate genomes -- abundant in higher primates, but present at much lower density in prosimians. Alu repeats are well-known for their capacity to promote non-allelic homologous recombination (NAHR) a process known to be inhibited by DNA-PK. Nanopore sequence analyses of cultured cells proficient or deficient in DNA-PK revealed an increase of inter-chromosomal translocations caused by NAHR. Although the high levels of DNA-PK in primates may have many functions, we posit that high levels of DNA-PK may function to restrain deleterious NAHR events between Alu elements.

Critical residues in the Ku70 von Willebrand A domain mediate Ku interaction with the LigIV-XRCC4 complex in non-homologous end-joining.

The Ku heterodimer (Ku70/Ku80) is central to the non-homologous end-joining (NHEJ) pathway. Ku binds to the broken DNA ends and promotes the assembly of the DNA repair complex. The N-terminal Ku70 von Willebrand A (vWA) domain is known to mediate protein-protein interactions important for the repair process. In particular, the D192 and D195 residues within helix 5 of the Ku70 vWA domain were shown to be essential for NHEJ function, although the precise role of these residues was not identified. Here, we set up a miniTurbo screening system to identify Ku70 D192/D195 residue-specific interactors in a conditional, human Ku70-knockout cell line in response to DNA damage. Using fusion protein constructs of Ku70 wild-type and mutant (D192A/D195R) with miniTurbo, we identified a number of candidate proximal interactors in response to DNA damage treatment, including DNA Ligase IV (LigIV), a known and essential NHEJ complex member. Interestingly, LigIV was enriched in our wildtype screen but not the Ku70 D192A/D195R screen, suggesting its interaction is disrupted by the mutation. Validation experiments demonstrated that the DNA damage-induced interaction between Ku70 and LigIV was disrupted by the Ku70 D192A/D195R mutations. Our findings provide greater detail about the interaction surface between the Ku70 vWA domain and LigIV and offer strong evidence that the D192 and D195 residues are important for NHEJ completion through an interaction with LigIV. Altogether, this work reveals novel potential proximal interactors of Ku in response to DNA damage and identifies Ku70 D192/D195 residues as essential for LigIV interaction with Ku during NHEJ.

In silico and in vitro characterization of the mycobacterial protein Ku to unravel its role in non-homologous end-joining DNA repair.

Non-homologous end-joining (NHEJ) stands as a pivotal DNA repair pathway crucial for the survival and persistence of Mycobacterium tuberculosis (Mtb) during its dormant, non-replicating phase, a key aspect of its long-term resilience. Mycobacterial NHEJ is a remarkably simple two-component system comprising the rate-limiting DNA binding protein Ku (mKu) and Ligase D. To elucidate mKu's role in NHEJ, we conducted a series of in silico and in vitro experiments. Molecular dynamics simulations and in vitro assays revealed that mKu's DNA binding stabilizes both the protein and DNA, while also shielding DNA ends from exonuclease degradation. Surface plasmon resonance (SPR) and electrophoretic mobility shift assays (EMSA) demonstrated mKu's robust affinity for linear double-stranded DNA (dsDNA), showing positive cooperativity for DNA substrates of 40 base pairs or longer, and its ability to slide along DNA strands. Moreover, analytical ultracentrifugation, size exclusion chromatography, and negative stain electron microscopy (EM) unveiled mKu's unique propensity to form higher-order oligomers exclusively with DNA, suggesting a potential role in mycobacterial NHEJ synapsis. This comprehensive characterization sheds new light on mKu's function within the Mtb NHEJ repair pathway. Targeting this pathway may thus impede the pathogen's ability to persist in its latent state within the host for prolonged periods.

Current status and prospect of the DNA double-strand break repair pathway in colorectal cancer development and treatment.

Colorectal cancer (CRC) is one of the most common malignancies worldwide. Double-strand break (DSB) is the most severe type of DNA damage. However, few reviews have thoroughly examined the involvement of DSB in CRC. Latest researches demonstrated that DSB repair plays an important role in CRC. For example, DSB-related genes such as BRCA1, Ku-70 and DNA polymerase theta (POLQ) are associated with the occurrence of CRC, and POLQ even showed to affect the prognosis and resistance for radiotherapy in CRC. This review comprehensively summarizes the DSB role in CRC, explores the mechanisms and discusses the association with CRC treatment. Four pathways for DSB have been demonstrated. 1. Nonhomologous end joining (NHEJ) is the major pathway. Its core genes including Ku70 and Ku80 bind to broken ends and recruit repair factors to form a complex that mediates the connection of DNA breaks. 2. Homologous recombination (HR) is another important pathway. Its key genes including BRCA1 and BRCA2 are involved in finding, pairing, and joining broken ends, and ensure the restoration of breaks in a normal double-stranded DNA structure. 3. Single-strand annealing (SSA) pathway, and 4. POLθ-mediated end-joining (alt-EJ) is a backup pathway. This paper elucidates roles of the DSB repair pathways in CRC, which could contribute to the development of potential new treatment approaches and provide new opportunities for CRC treatment and more individualized treatment options based on therapeutic strategies targeting these DNA repair pathways.

m6A-mediated lnc-OXAR promotes oxaliplatin resistance by enhancing Ku70 stability in non-alcoholic steatohepatitis-related hepatocellular carcinoma.

BACKGROUND: The escalating prevalence of metabolic diseases has led to a rapid increase in non-alcoholic steatohepatitis (NASH)-related hepatocellular carcinoma (NASH-HCC). While oxaliplatin (OXA)-based hepatic arterial infusion chemotherapy (HAIC) has shown promise in advanced-stage HCC patients, its efficacy in NASH-HCC remains uncertain. This study aims to assess the effectiveness of OXA-based HAIC and elucidate the mechanisms underlying OXA resistance in NASH-HCC. METHODS: The key lncRNAs were screened through RNA-seq analysis of NASH/non-NASH and OXA-sensitive/OXA-resistant (OXA-S/R) HCC tissues. The biological functions of the lnc-OXAR (OXA resistance-related lncRNA in NASH-HCC) in NASH-HCC were verified through a series of in vitro and in vivo experiments. The molecular mechanism of lnc-OXAR was elucidated by fluorescence in situ hybridization, immunoprecipitation-mass spectrometry (FISH), Immunoprecipitation-Mass Spectrometry (IP-MS), RNA pulldown, RNA immunoprecipitation (RIP), methylated RNA immunoprecipitation sequencing (MeRIP-Seq) and a dual-luciferase reporter assay. RESULTS: NASH-HCC exhibited reduced responsiveness to OXA-based HAIC compared to non-NASH HCC. We identified and validated a novel transcript namedlnc-OXAR, which played a crucial role in conferring OXA resistance to NASH-HCC. Inhibition of lnc-OXAR suppressed HCC cell growth and restored OXA sensitivity both in NASH-HCC mouse models and in vitro. Mechanistically, lnc-OXAR recruited Ku70 and cystatin A (CSTA), preventing Ku70 degradation and facilitating DNA double-strand break (DSB) repair, thereby promoting OXA resistance in NASH-HCC. Additionally, WTAP-mediated m6A modification enhanced the stability of lnc-OXAR in an IGF2BP2-dependent manner. Notably, silencing lnc-OXAR significantly enhanced the response to OXA in patient-derived xenograft (PDX) models derived from NASH-HCC. CONCLUSIONS: The reduced responsiveness of NASH-HCC to OXA treatment can be attributed to the upregulation of lnc-OXAR. Our findings provide a rationale for stratifying HCC patients undergoing OXA-based HAIC based on etiology. Lnc-OXAR holds promise as a novel target for overcoming OXA resistance in NASH-HCC and improving prognosis.

Anti-Ku + myositis: an acquired inflammatory protein-aggregate myopathy.

Myositis with anti-Ku-autoantibodies is a rare inflammatory myopathy associated with various connective tissue diseases. Histopathological studies have identified inflammatory and necrotizing aspects, but a precise morphological analysis and pathomechanistic disease model are lacking. We therefore aimed to carry out an in-depth morpho-molecular analysis to uncover possible pathomechanisms. Muscle biopsy specimens from 26 patients with anti-Ku-antibodies and unequivocal myositis were analyzed by immunohistochemistry, immunofluorescence, transcriptomics, and proteomics and compared to biopsy specimens of non-disease controls, immune-mediated necrotizing myopathy (IMNM), and inclusion body myositis (IBM). Clinical findings and laboratory parameters were evaluated retrospectively and correlated with morphological and molecular features. Patients were mainly female (92%) with a median age of 56.5 years. Isolated myositis and overlap with systemic sclerosis were reported in 31%, respectively. Isolated myositis presented with higher creatine kinase levels and cardiac involvement (83%), whereas systemic sclerosis-overlap patients often had interstitial lung disease (57%). Histopathology showed a wide spectrum from mild to pronounced myositis with diffuse sarcolemmal MHC-class I (100%) and -II (69%) immunoreactivity, myofiber necrosis (88%), endomysial inflammation (85%), thickened capillaries (84%), and vacuoles (60%). Conspicuous sarcoplasmic protein aggregates were p62, BAG3, myotilin, or immunoproteasomal beta5i-positive. Proteomic and transcriptomic analysis identified prominent up-regulation of autophagy, proteasome, and hnRNP-related cell stress. To conclude, Ku + myositis is morphologically characterized by myofiber necrosis, MHC-class I and II positivity, variable endomysial inflammation, and distinct protein aggregation varying from IBM and IMNM, and it can be placed in the spectrum of scleromyositis and overlap myositis. It features characteristic sarcoplasmic protein aggregation on an acquired basis being functionally associated with altered chaperone, proteasome, and autophagy function indicating that Ku + myositis exhibit aspects of an acquired inflammatory protein-aggregate myopathy.

LINC-PINT plays an anti-tumor role in nasopharyngeal carcinoma by binding to XRCC6 and affecting its function.

BACKGROUND: LINC-PINT was downregulated in nasopharyngeal carcinoma (NPC) and correlated with treatment efficiency of NPC. However, the underlying mechanism of LINC-PINT in NPC has not yet been fully explored. METHOD: We used CellTiter luminescent assay, clone formation assay, Hoechst staining, and SYTO-9/PI staining to examine cell viability and cell apoptosis regulated by LINC-PINT in NPC cells. Xenograft tumor model, HE staining, Ki67 staining, and TUNEL assay were conducted to assess the role of LINC-PINT in vivo. Bioinformatics and RNA immunoprecipitation assay was performed to identify the binding protein of LINC-PINT. Fluorescence in situ hybridization and immunofluorescence were utilized to measure the colocalization of XRCC6 with LINC-PINT and DNA-PKcs. Mito-Tracker red CMXRos staining was used to label mitochondria in cells specifically. RESULT: We found LINC-PINT was downregulated in many tumors (including NPC) and associated with poor prognosis. The cell viability was significantly inhibited and cell apoptosis was remarkably promoted in LINC-PINT overexpressed cells in contrast to control cells. The growth of tumor xenografts was significantly suppressed and the tumor weight was significantly decreased in LINC-PINT overexpression group compared to the control group. Correspondingly, the positive Ki67 foci was decreased while TUNEL foci was increased in LINC-PINT overexpression group. Mechanically, we verified XRCC6 as a new binding protein of LINC-PINT through RNA binding domains prediction, RIP and colocalization of LINC-PINT and XRCC6. By binding to XRCC6, LINC-PINT interfered the formation of DNA-PK complex, regulated mitochondria accumulation status and affected the modification of apoptosis proteins, leading to more cell apoptosis. CONCLUSION: Our study provided the first evidence that LINC-PINT promotes cell apoptosis in NPC by binding to XRCC6 and affecting its function.

The DNA double-strand break repair proteins γH2AX, RAD51, BRCA1, RPA70, KU80, and XRCC4 exhibit follicle-specific expression differences in the postnatal mouse ovaries from early to older ages.

PURPOSE: Ovarian aging is closely related to a decrease in follicular reserve and oocyte quality. The precise molecular mechanisms underlying these reductions have yet to be fully elucidated. Herein, we examine spatiotemporal distribution of key proteins responsible for DNA double-strand break (DSB) repair in ovaries from early to older ages. Functional studies have shown that the γH2AX, RAD51, BRCA1, and RPA70 proteins play indispensable roles in HR-based repair pathway, while the KU80 and XRCC4 proteins are essential for successfully operating cNHEJ pathway. METHODS: Female Balb/C mice were divided into five groups as follows: Prepuberty (3 weeks old; n = 6), puberty (7 weeks old; n = 7), postpuberty (18 weeks old; n = 7), early aged (52 weeks old; n = 7), and late aged (60 weeks old; n = 7). The expression of DSB repair proteins, cellular senescence (ß-GAL) and apoptosis (cCASP3) markers was evaluated in the ovaries using immunohistochemistry. RESULT: ß-GAL and cCASP3 levels progressively increased from prepuberty to aged groups (P < 0.05). Notably, γH2AX levels varied in preantral and antral follicles among the groups (P < 0.05). In aged groups, RAD51, BRCA1, KU80, and XRCC4 levels increased (P < 0.05), while RPA70 levels decreased (P < 0.05) compared to the other groups. CONCLUSIONS: The observed alterations were primarily attributed to altered expression in oocytes and granulosa cells of the follicles and other ovarian cells. As a result, the findings indicate that these DSB repair proteins may play a role in the repair processes and even other related cellular events in ovarian cells from early to older ages.

Identification of the main barriers to Ku accumulation in chromatin.

Repair of DNA double-strand breaks by the non-homologous end-joining pathway is initiated by the binding of Ku to DNA ends. Multiple Ku proteins load onto linear DNAs in vitro. However, in cells, Ku loading is limited to ∼1-2 molecules per DNA end. The mechanisms enforcing this limit are currently unclear. Here, we show that the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), but not its protein kinase activity, is required to prevent excessive Ku entry into chromatin. Ku accumulation is further restricted by two mechanisms: a neddylation/FBXL12-dependent process that actively removes loaded Ku molecules throughout the cell cycle and a CtIP/ATM-dependent mechanism that operates in S phase. Finally, we demonstrate that the misregulation of Ku loading leads to impaired transcription in the vicinity of DNA ends. Together, our data shed light on the multiple mechanisms operating to prevent Ku from invading chromatin and interfering with other DNA transactions.

[The Identification Idea of Antibodies Against Ku and Other High-Frequency Antigens].

OBJECTIVE: This study was aimed to provide ideas for identifying the antibodies to high-frequency antigens by analyzing a female case of high-frequency antigen antibody (anti-Ku) using serological and sequencing method. METHODS: The methods for identification of blood group, erythrocyte antigen, screening and identification of antibody were used to detect the blood type and antibody in the proband. The proband's serum and reagent screening cells treated with Sulfhydryl reagent were applied to judge the type and characteristics of this antibodies when reacted with the regaent screening cells or proband's serum respectively. Gene sequencing was used to determine the genotype of the proband's blood group. RESULTS: The proband's red blood cells were determined as O type RhD positive, whose serum showed strong positive reaction to antibody-screening cells and antibody identification cells with the same intensity in saline and IAT medium, however, the self-cells showed negative effect. The Direct Antihuman Globulin of proband's red blood cells also showed weak positive reaction, and the other blood types were CcEe, Jk(a+b-), P1-, Le(a-b -), Lu (a-b +), K-, k-, Kp(a-b-). Serum of the proband treated with 2-ME still react with three groups of screening cells in IAT medium. The reaction intensity of proband's serum was also unchanged with the cells modified with papain and bromelain, but showed negative effect when the cells were treated with sulfhydryl agents including DTT and 2-ME. Gene sequencing revealed that the KEL genotype of the patient was KEL*02N.24 . This patient had a rare K0 phenotype. CONCLUSION: The rare Kell-null blood group (also known as K0) were identified by serological and molecular tests in the proband who produced both IgG and IgM type of antibody to high-frequency antigen (anti-Ku). These two methods are of great significance in the identification of this rare blood group as well as the antibody to high frequency antigen.

Characterization of a KU70-disrupted strain of the mannosylerythritol lipid-producing yeast Pseudozyma tsukubaensis constructed by a marker recycling system.

The basidiomycetous yeast Pseudozyma tsukubaensis is known as an industrial mannosylerythritol lipid producer. In this study, the PtURA5 marker gene was deleted by homologous recombination. Using the PtURA5-deleted mutant as a host strain, we obtained a derivative disrupted for the PtKU70 gene, a putative ortholog of the KU70 gene encoding a protein involved in the nonhomologous end-joining pathway of DNA repair. Subsequently, the introduced PtURA5 gene was re-deleted by marker recycling. These results demonstrated that the PtURA5 gene can be used as a recyclable marker gene. Although the frequency of homologous recombination has been shown to be increased by KU70 disruption in other fungi, the PtKU70-disrupted strain of P. tsukubaensis did not demonstrate an elevated frequency of homologous recombination. Furthermore, the PtKU70-disrupted strain did not show increased susceptibility to bleomycin. These results suggested that the function of this KU70 ortholog in P. tsukubaensis is distinct from that in other fungi.

KBM-mediated interactions with KU80 promote cellular resistance to DNA replication stress in CHO cells.

The KU heterodimer (KU70/80) is rapidly recruited to DNA double-strand breaks (DSBs) to regulate their processing and repair. Previous work has revealed that the amino-terminal von Willebrand-like (vWA-like) domain in KU80 harbours a conserved hydrophobic pocket that interacts with a short peptide motif known as the Ku-binding motif (KBM). The KBM is present in a variety of DNA repair proteins such as APLF, CYREN, and Werner protein (WRN). Here, to investigate the importance of KBM-mediated protein-protein interactions for KU80 function, we employed KU80-deficient Chinese Hamster Ovary (Xrs-6) cells transfected with RFP-tagged wild-type human KU80 or KU80 harbouring a mutant vWA-like domain (KU80L68R). Surprisingly, while mutant RFP-KU80L68R largely or entirely restored NHEJ efficiency and radiation resistance in KU80-deficient Xrs-6 cells, it failed to restore cellular resistance to DNA replication stress induced by camptothecin (CPT) or hydroxyurea (HU). Moreover, KU80-deficient Xrs-6 cells expressing RFP-KU80L68R accumulated pan-nuclear γH2AX in an S/G2-phase-dependent manner following treatment with CPT or HU, suggesting that the binding of KU80 to one or more KBM-containing proteins is required for the processing and/or repair of DNA ends that arise during DNA replication stress. Consistent with this idea, depletion of WRN helicase/exonuclease recapitulated the CPT-induced γH2AX phenotype, and did so epistatically with mutation of the KU80 vWA-like domain. These data identify a role for the KBM-binding by KU80 in the response and resistance of CHO cells to arrested and/or collapsed DNA replication forks, and implicate the KBM-mediated interaction of KU80 with WRN as a critical effector of this role.

KDM6A-SND1 interaction maintains genomic stability by protecting the nascent DNA and contributes to cancer chemoresistance.

Genomic instability is one of the hallmarks of cancer. While loss of histone demethylase KDM6A increases the risk of tumorigenesis, its specific role in maintaining genomic stability remains poorly understood. Here, we propose a mechanism in which KDM6A maintains genomic stability independently on its demethylase activity. This occurs through its interaction with SND1, resulting in the establishment of a protective chromatin state that prevents replication fork collapse by recruiting of RPA and Ku70 to nascent DNA strand. Notably, KDM6A-SND1 interaction is up-regulated by KDM6A SUMOylation, while KDM6AK90A mutation almost abolish the interaction. Loss of KDM6A or SND1 leads to increased enrichment of H3K9ac and H4K8ac but attenuates the enrichment of Ku70 and H3K4me3 at nascent DNA strand. This subsequently results in enhanced cellular sensitivity to genotoxins and genomic instability. Consistent with these findings, knockdown of KDM6A and SND1 in esophageal squamous cell carcinoma (ESCC) cells increases genotoxin sensitivity. Intriguingly, KDM6A H101D & P110S, N1156T and D1216N mutations identified in ESCC patients promote genotoxin resistance via increased SND1 association. Our finding provides novel insights into the pivotal role of KDM6A-SND1 in genomic stability and chemoresistance, implying that targeting KDM6A and/or its interaction with SND1 may be a promising strategy to overcome the chemoresistance.

Ku70 Binding to YAP Alters PARP1 Ubiquitination to Regulate Genome Stability and Tumorigenesis.

Yes-associated protein (YAP) is a central player in cancer development, with functions extending beyond its recognized role in cell growth regulation. Recent work has identified a link between YAP/transcriptional coactivator with PDZ-binding motif (TAZ) and the DNA damage response. Here, we investigated the mechanistic underpinnings of the cross-talk between DNA damage repair and YAP activity. Ku70, a key component of the nonhomologous end joining pathway to repair DNA damage, engaged in a dynamic competition with TEAD4 for binding to YAP, limiting the transcriptional activity of YAP. Depletion of Ku70 enhanced interaction between YAP and TEAD4 and boosted YAP transcriptional capacity. Consequently, Ku70 loss enhanced tumorigenesis in colon cancer and hepatocellular carcinoma (HCC) in vivo. YAP impeded DNA damage repair and elevated genome instability by inducing PARP1 degradation through the SMURF2-mediated ubiquitin-proteasome pathway. Analysis of samples from patients with HCC substantiated the link between Ku70 expression, YAP activity, PARP1 levels, and genome instability. In conclusion, this research provides insight into the mechanistic interactions between YAP and key regulators of DNA damage repair, highlighting the role of a Ku70-YAP-PARP1 axis in preserving genome stability. Significance: Increased yes-associated protein transcriptional activity stimulated by loss of Ku70 induces PARP1 degradation by upregulating SMURF2 to inhibit DNA damage, driving genome instability and tumorigenesis.