Impact of Optimized Ku-DNA Binding Inhibitors on the Cellular and In Vivo DNA Damage Response.

Background: DNA-dependent protein kinase (DNA-PK) is a validated cancer therapeutic target involved in DNA damage response (DDR) and non-homologous end-joining (NHEJ) repair of DNA double-strand breaks (DSBs). Ku serves as a sensor of DSBs by binding to DNA ends and activating DNA-PK. Inhibition of DNA-PK is a common strategy to block DSB repair and improve efficacy of ionizing radiation (IR) therapy and radiomimetic drug therapies. We have previously developed Ku-DNA binding inhibitors (Ku-DBis) that block in vitro and cellular NHEJ activity, abrogate DNA-PK autophosphorylation, and potentiate cellular sensitivity to IR. Results and Conclusions: Here we report the discovery of oxindole Ku-DBis with improved cellular uptake and retained potent Ku-inhibitory activity. Variable monotherapy activity was observed in a panel of non-small cell lung cancer (NSCLC) cell lines, with ATM-null cells being the most sensitive and showing synergy with IR. BRCA1-deficient cells were resistant to single-agent treatment and antagonistic when combined with DSB-generating therapies. In vivo studies in an NSCLC xenograft model demonstrated that the Ku-DBi treatment blocked IR-dependent DNA-PKcs autophosphorylation, modulated DDR, and reduced tumor cell proliferation. This represents the first in vivo demonstration of a Ku-targeted DNA-binding inhibitor impacting IR response and highlights the potential therapeutic utility of Ku-DBis for cancer treatment.

Design and architecting of a broadband bullet-diamond shaped integrated frequency selective meta-surface absorber for aero-stealth platform.

The work report on architecture of integrated frequency selective meta-surface (IFSMS) absorbers for aerospace stealth applications. Fabricated IFSMS comprised of a pattern metasurface integrated with dielectric interlayer and conducting ground. Initially, a supercell (2 × 2-unit cell: 24 × 24 mm2) was designed with a fourfold topological symmetry. Supercell produces impedances (R), inductances (L), and capacitances (C) in tune with design on its interaction with microwave. RC performance was tested at variable incident transverse electric/magnetic (TE/TM) modes over, Θ, 0°-60° and at the normal incidence (TE), against a planer, clockwise rotation over, Φ, 0°-90°. The mode stability and rotational invariance was analyzed for displacement current- and power-density distributions. The impedance behavior and phase reversal S11 reflection coefficient studies revealed the emergence of mid-band Fabry-Perot mode distinguishing LC behavior of the circuit. The meta-pattern was manufactured by mask lithography using a customized resistive micro-carbon ink and imprinted onto dielectric/ground tile (dimension: 30 × 30 cm2). Structure-property relationship of the ink material was investigated using SEM, XRD, FTIR, UV-visible spectroscopy to reveled surface properties of imprinted material. The absorber was subjected to the free space measurements over C (4-8), X (8-12), and Ku (12-18 GHz) bands, including pristine interlayer dielectrics. The simulated and experimental RC data was found to be in excellent agreement. The proposed IFSMS design is a potential candidate for the stealth application.

Integrating Kalman filter noise residue into U-Net for robust image denoising: the KU-Net model.

In low-level image processing, where the main goal is to reconstruct a clean image from a noise-corrupted version, image denoising continues to be a critical challenge. Although recent developments have led to the introduction of complex architectures to improve denoising performance, these models frequently have more parameters and higher computational demands. Here, we propose a new, simplified architecture called KU-Net, which is intended to achieve better denoising performance while requiring less complexity. KU-Net is an extension of the basic U-Net architecture that incorporates gradient information and noise residue from a Kalman filter. The network's ability to learn is improved by this deliberate incorporation, which also helps it better preserve minute details in the denoised images. Without using Image augmentation, the proposed model is trained on a limited dataset to show its resilience in restricted training settings. Three essential inputs are processed by the architecture: gradient estimations, the predicted noisy image, and the original noisy grey image. These inputs work together to steer the U-Net's encoding and decoding stages to generate high-quality denoised outputs. According to our experimental results, KU-Net performs better than traditional models, as demonstrated by its superiority on common metrics like the Structural Similarity Index (SSIM) and Peak Signal-to-Noise Ratio (PSNR). KU-Net notably attains a PSNR of 26.60 dB at a noise level of 50, highlighting its efficacy and potential for more widespread use in image denoising.

Tissue microarray analyses of the essential DNA repair factors ATM, DNA-PKcs and Ku80 in head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) negative for Human Papillomavirus (HPV) has remained a difficult to treat entity, whereas tumors positive for HPV are characterized by radiosensitivity and favorable patient outcome. On the cellular level, radiosensitivity is largely governed by the tumor cells` ability to repair radiation-induced DNA double-strand breaks (DSBs), but no biomarker is established that could guide clinical decision making. Therefore, we tested the impact of the expression levels of ATM, the central kinase of the DNA damage response as well as DNA-PKcs and Ku80, two major factors in the main DSB repair pathway non-homologous end joining (NHEJ). METHODS: A tissue microarray of a single center HNSCC cohort was stained for ATM, DNA-PKcs and Ku80 and the expression scored based on staining intensity and the percentages of tumor cells stained. Scores were correlated with clinicopathological parameters and survival. RESULTS: Samples from 427 HNSCC patients yielded interpretable stainings and were scored following an established algorithm. The majority of tumors showed strong expression of both NHEJ factors, whereas the expression of ATM varied more. The expression scores of ATM and DNA-PKcs were not associated with patient survival. For HPV-negative HNSCC, the minority of tumors without strong Ku80 expression trended towards superior survival when treatment included radiotherapy. Focusing stronger on staining intensity to define the subgroup with lowest and therefore potentially insufficient expression levels in the HPV-negative subgroup, we observed significantly better overall survival for patients treated with radiotherapy but not with surgery alone. CONCLUSIONS: Our data suggest that HPV-negative HNSCC with particularly low Ku80 expression represent a highly radiosensitive subpopulation. Confirmation in independent cohorts is required.

Not Just Myocarditis: Mixed Connective Tissue Disease (MCTD) and Overlap Myositis With Anti-Ku Positivity in a Young Male With Shortness of Breath.

Mixed connective tissue disease (MCTD) is an autoimmune disorder characterized by high levels of anti-U1 ribonucleoprotein (RNP) antibodies and overlapping clinical features of autoimmune diseases, such as systemic lupus erythematosus (SLE), systemic sclerosis (SSc), and polymyositis (PM). Anti-Ku antibodies have been associated with overlap syndromes, which can present with symptoms such as Raynaud's phenomenon, arthritis, and myositis.  A 19-year-old male athlete presented with myositis, notable for cardiac involvement. Diagnostic testing revealed elevated anti-RNP and anti-Ku antibodies, and a muscle biopsy indicated scleromyositis/overlap myositis. The patient was treated with high-dose corticosteroids, intravenous immunoglobulin (IVIG), rituximab, and mycophenolate mofetil, which led to significant improvement in muscle strength and cardiac function. This case highlights the diagnostic complexity of MCTD when associated with positive anti-Ku antibodies, overlap syndromes, and cardiac involvement. Successful management emphasizes the importance of a tailored, multi-modal therapeutic approach.

Design and On-Orbit Performance of Ku-Band Phased-Array Synthetic-Aperture Radar Payload System.

The current emphasis in the advancement of space-based synthetic-aperture radar (SAR) is on lightweight payloads under 100 kg with resolutions surpassing 1 m. This focus is directed toward meeting the launch criteria for multiple satellites on a single rocket and cutting costs. This article discusses the creation and progress of a Ku-band SAR payload for the Taijing-4(03) satellite, launched on 23 January 2024 and accompanied by four other satellites. The SAR payload design was customized to meet the demands of a micro-nano satellite platform, resulting in a lightweight, flat design weighing less than 80 kg, seamlessly integrated with the plate-shaped satellite platform. The article also introduces a beam optimization strategy for the phased array SAR antenna, significantly boosting the SAR system's performance. The SAR payload provides various operating modes like slide-spot, strip, Scan 1, Scan 2, and others, with a maximum achievable resolution exceeding 1 m. Extensive in-orbit testing of the payload produced numerous high-quality SAR images with potential uses in emergency disaster mitigation, safeguarding ecosystems, monitoring forests, managing crops, tracking sea ice, and more.

A challenging case of hemolytic disease of the fetus and newborn (HDFN) due to anti-Ku in a K0 (Kellnull) mother.

Hemolytic disease of the fetus and newborn (HDFN) due to an antibody in the Kell blood group system can be associated with severe fetal anemia. This case report details the challenges of managing a Kellnull mother with anti-Ku that affected her fetus/newborn. A gravida 4 para 3 woman at term underwent an emergency lower caesarean section because of fetal distress. The baby was intubated because of low oxygen saturation. An urgent request for a hematology workup showed severe anemia and erythroblastosis fetalis. Unfortunately, no compatible blood was found, and the baby died. The case was referred to the National Blood Centre, and anti-Ku was confirmed in a sample sent from the mother. When she presented with her fifth pregnancy, meticulous planning was used to manage this pregnancy. Her family screening revealed one brother with a matching phenotype. Three blood donations were planned for the brother-for freezing, for intrauterine transfusion, and for standby during delivery. Serial anti-Ku titrations of maternal samples were performed, and the fetus was monitored for anemia through middle cerebral artery Doppler scans. Although the anti-Ku titers reached as high as 1024, fetal anemia was never diagnosed. The neonate was delivered safely but was diagnosed with severe pathologic jaundice and anemia secondary to HDFN and congenital pneumonia. The baby was transfused with K0 packed red blood cells and later discharged to home.

Tyrosyl-DNA phosphodiesterase 2 (Tdp2) repairs DNA-protein crosslinks and protects against double strand breaks in vivo.

DNA-protein crosslinks pose a significant challenge to genome stability and cell viability. Efficient repair of DPCs is crucial for preserving genomic integrity and preventing the accumulation of DNA damage. Despite recent advances in our understanding of DPC repair, many aspects of this process, especially at the organismal level, remain elusive. In this study, we used zebrafish as a model organism to investigate the role of TDP2 (Tyrosyl-DNA phosphodiesterase 2) in DPC repair. We characterized the two tdp2 orthologs in zebrafish using phylogenetic, syntenic and expression analysis and investigated the phenotypic consequences of tdp2 silencing in zebrafish embryos. We then quantified the effects of tdp2a and tdp2b silencing on cellular DPC levels and DSB accumulation in zebrafish embryos. Our findings revealed that tdp2b is the main ortholog during embryonic development, while both orthologs are ubiquitously present in adult tissues. Notably, the tdp2b ortholog is phylogenetically closer to human TDP2. Silencing of tdp2b, but not tdp2a, resulted in the loss of Tdp2 activity in zebrafish embryos, accompanied by the accumulation of DPCs and DSBs. Our findings contribute to a more comprehensive understanding of DPC repair at the organismal level and underscore the significance of TDP2 in maintaining genome stability.

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.

Enhancing surgical precision: a novel electromyography finding for confident identification of the root exit zone during microvascular decompression surgery.

OBJECTIVE: The lateral spread response (LSR) is an important electrophysiological sign that predicts successful decompression in patients undergoing microvascular decompression (MVD) for hemifacial spasm (HFS). However, LSRs do not consistently correlate with clinical outcomes, and there are cases in which LSRs are absent. In this study, the authors identified a unique pattern on facial nerve electromyography (EMG) when the root exit zone (REZ) is touched. This distinctive EMG pattern, which the authors coined the "Sang-ku sign" (SKS), could aid in identifying the offending vessel on the REZ, even in the absence of LSRs. METHODS: Between November 2022 and August 2023, the authors analyzed EMG findings from 185 patients undergoing MVD for HFS. Bipolar stimulation electrodes were placed at the marginal mandibular branch, and recordings were obtained from the frontalis and orbicularis oris muscles. Additionally, stimulation electrodes were placed at the temporal branch, and recordings were obtained from the oris and mentalis muscles. The authors statistically analyzed the presence of SKS and its association with demographic, surgical, clinical outcomes, and anatomical factors. RESULTS: The authors observed a brief, burst-like wave pattern arising from all recorded muscle branches when Teflon felt was placed between the REZ and the facial nerve. This EMG pattern, the SKS, was found in 164 patients (88.6%). Among the factors analyzed, only patient age showed a significant association with the presence of the SKS in univariate analysis (p = 0.007). The presence of the SKS was significantly associated with predicting the disappearance of LSRs (p = 0.045). Clinical outcomes were favorable (99% at the last follow-up) in all cases; thus, no positive correlation was observed in the existence of the SKS and LSRs. CONCLUSIONS: The SKS could serve as an excellent guide for the facial nerve REZ during surgery. Given that HFS results from abnormal vascular contact on the REZ, this finding plays a crucial role in ensuring surgical success. Alongside LSRs, the SKS could provide valuable insights for neurosurgeons.

Efficient RNA interference method by feeding in Brachionus plicatilis (Rotifera).

Rotifers are small, ubiquitous invertebrate animals found throughout the world and have emerged as a promising model system for studying molecular mechanisms in the fields of experimental ecology, aquatic toxicology, and geroscience. However, the lack of efficient gene expression manipulation techniques has hindered the study of rotifers. In this study, we used the L4440 plasmid with two reverse-oriented T7 promoters, along with RNase-deficient E. coli HT115, to efficiently produce dsRNA and thereby present an efficient feeding-based RNAi method in Brachionus plicatilis. We targeted Bp-Ku70 & Ku80, key proteins in the DNA double-strand breaks repair pathway, and then subjected rotifers to UV radiation. We found that the mRNA expression, fecundity, as well as survival rate diminished significantly as a result of RNAi. Overall, our results demonstrate that the feeding-based RNAi method is a simple and efficient tool for gene knockdown in B. plicatilis, advancing their use as a model organism for biological research.

Oligonucleotide-based CRISPR-Cas9 toolbox for efficient engineering of Komagataella phaffii.

Komagataella phaffii (Pichia pastoris) is a methylotrophic yeast that is favored by industry and academia mainly for expression of heterologous proteins. However, its full potential as a host for bioproduction of valuable compounds cannot be fully exploited as genetic tools are lagging behind those that are available for baker's yeast. The emergence of CRISPR-Cas9 technology has significantly improved the efficiency of gene manipulations of K. phaffii, but improvements in gene-editing methods are desirable to further accelerate engineering of this yeast. In this study, we have developed a versatile vector-based CRISPR-Cas9 method and showed that it works efficiently at different genetic loci using linear DNA fragments with very short targeting sequences including single-stranded oligonucleotides. Notably, we performed site-specific point mutations and full gene deletions using short (90 nt) single-stranded oligonucleotides at very high efficiencies. Lastly, we present a strategy for transient inactivation of nonhomologous end-joining (NHEJ) pathway, where KU70 gene is disrupted by a visual marker (uidA gene). This system enables precise CRISPR-Cas9-based editing (including multiplexing) and facilitates simple reversion to NHEJ-proficient genotype. In conclusion, the tools presented in this study can be applied for easy and efficient engineering of K. phaffii strains and are compatible with high-throughput automated workflows.

An efficient gene targeting system using Δku80 and functional analysis of Cyp51A in Trichophyton rubrum.

Trichophyton rubrum is one of the most frequently isolated fungi in patients with dermatophytosis. Despite its clinical significance, the molecular mechanisms of drug resistance and pathogenicity of T. rubrum remain to be elucidated because of the lack of genetic tools, such as efficient gene targeting systems. In this study, we generated a T. rubrum strain that lacks the nonhomologous end-joining-related gene ku80 (Δku80) and then developed a highly efficient genetic recombination system with gene targeting efficiency that was 46 times higher than that using the wild-type strain. Cyp51A and Cyp51B are 14-α-lanosterol demethylase isozymes in T. rubrum that promote ergosterol biosynthesis and are the targets of azole antifungal drugs. The expression of cyp51A mRNA was induced by the addition of the azole antifungal drug efinaconazole, whereas no such induction was detected for cyp51B, suggesting that Cyp51A functions as an azole-responsive Cyp51 isozyme. To explore the contribution of Cyp51A to susceptibility to azole drugs, the neomycin phosphotransferase (nptII) gene cassette was inserted into the cyp51A 3'-untranslated region of Δku80 to destabilize the mRNA of cyp51A. In this mutant, the induction of cyp51A mRNA expression by efinaconazole was diminished. The minimum inhibitory concentration for several azole drugs of this strain was reduced, suggesting that dermatophyte Cyp51A contributes to the tolerance for azole drugs. These findings suggest that an efficient gene targeting system using Δku80 in T. rubrum is applicable for analyzing genes encoding drug targets.

Digital Reconstruction: A Critical Examination of the History and Adaptation of Ku Klux Klan Websites.

In response to the data revolution, academic research and media attention have increasingly focused on the technological adaptation and innovation displayed by the far right. The greatest attention is paid to social media and how groups and organizations are utilizing technological advancement and growth in virtual networks to increase recruitment and advance radicalization on a global scale. As with most social and political endeavors, certain technologies are in vogue and thus draw the attention of users and regulators and service providers. This creates a technological blind spot within which extremist groups frequently operate older and less well regarded technologies without the oversight that one might expect. This article examines the less well-studied traditional and official websites of the Ku Klux Klan, the most established and iconic of American far-right organizations. By incorporating non-participant observation of online spaces and thematic analysis, this research analyzes the evolution of 26 websites, from their emergence in the early 1990s to the present day. We examine the ways in which traditional printed communications and other ephemera have progressed with advances in technology, focusing on the following central elements of Klan political activism and community formation: Klan identity, organizational history, aims and objectives; technology and outreach, including online merchandise and event organization; and the constructions of whiteness and racism. The results add value and insight to comparable work by offering a unique historical insight into the ways in which the Klan have developed and made use of Web 1.0, Web 2.0, and Web3 technologies.

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.

Biofortification, metabolomic profiling and quantitative analysis of vitamin B12 enrichment in guava juice via lactic acid fermentation using Levilactobacillus brevis strain KU15152.

BACKGROUND: Chemical fortification and dose supplementation of vitamin B12 are widely implemented to combat deficiency symptoms. However, in situ, fortification of vitamin B12 in food matrixes can be a promising alternative to chemical fortification. The present study aimed to produce vitamin B12-rich, probiotic guava juice fermented with Levilactobacillus brevis strain KU15152. Pasteurized fresh guava juice was inoculated with 7.2 log CFU mL-1 L. brevis strain KU15152 and incubated for 72 h at 37 °C anaerobically. The antioxidants, total phenolic compounds, vitamin B12 production, sugars, organic acids, pH and viable count were analyzed at 24, 48 and 72 h of incubation. The fermented juice was stored at 4 °C, and the changes in its functional properties were analyzed at 7-day intervals up to 28 days of storage. RESULTS: During fermentation, the bacteria cell count was increased from 7.01 ± 0.06 to 9.76 ± 0.42 log CFU mL-1 after 72 h of fermentation and was decreased to 6.94 ± 0.34 CFU mL-1 during storage at 4 °C after 28 days. The pH, total soluble solids, crude fiber, citric acid and total sugars decreased, while titratable acidity, total protein, antioxidants, phenolic compounds and lactic acid contents increased during fermentation. The fermented guava juice exhibited higher 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS)) radical scavenging activities (85.97% and 75.97%, respectively) at 48 h of fermentation. The concentration of active vitamin B12 in the sample reached 109.5 µg L-1 at 72 h of fermentation. However, this concentration gradually decreased to 70.2 µg L-1 during the storage period. During storage for 28 days at 4 °C, both the fermented and control guava juices exhibited a decline in antioxidant and phenolic compound concentrations. Furthermore, the addition of 20% honey and guava flavor enhanced the organoleptic properties and acceptability of fermented guava juice. CONCLUSION: The value-added fermented guava juice could be a novel functional food product to combat vitamin B12 deficiency. © 2024 The Author(s). Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

[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.

Di- and tri-methylation of histone H3K36 play distinct roles in DNA double-strand break repair.

Histone H3 Lys36 (H3K36) methylation and its associated modifiers are crucial for DNA double-strand break (DSB) repair, but the mechanism governing whether and how different H3K36 methylation forms impact repair pathways is unclear. Here, we unveil the distinct roles of H3K36 dimethylation (H3K36me2) and H3K36 trimethylation (H3K36me3) in DSB repair via non-homologous end joining (NHEJ) or homologous recombination (HR). Yeast cells lacking H3K36me2 or H3K36me3 exhibit reduced NHEJ or HR efficiency. yKu70 and Rfa1 bind H3K36me2- or H3K36me3-modified peptides and chromatin, respectively. Disrupting these interactions impairs yKu70 and Rfa1 recruitment to damaged H3K36me2- or H3K36me3-rich loci, increasing DNA damage sensitivity and decreasing repair efficiency. Conversely, H3K36me2-enriched intergenic regions and H3K36me3-enriched gene bodies independently recruit yKu70 or Rfa1 under DSB stress. Importantly, human KU70 and RPA1, the homologs of yKu70 and Rfa1, exclusively associate with H3K36me2 and H3K36me3 in a conserved manner. These findings provide valuable insights into how H3K36me2 and H3K36me3 regulate distinct DSB repair pathways, highlighting H3K36 methylation as a critical element in the choice of DSB repair pathway.

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.