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Anti‐cancer properties of (‐)‐epigallocatechin‐3‐gallate (EGCG) are mediated via apoptosis induction, as well as inhibition of cell proliferation and histone deacetylase. Accumulation of stabilized cellular FLICE‐inhibitory protein (c‐FLIP)/Ku70 complex in the cytoplasm inhibits apoptosis through interruption of extrinsic apoptosis pathway. In this study, we evaluated the anti‐cancer role of EGCG in gastric cancer (GC) cells through dissociation of c‐FLIP/Ku70 complex. MKN‐45 cells were treated with EGCG or its antagonist MG149 for 24 h. Apoptosis was evaluated by flow cytometry and quantitative RT‐PCR. Protein expression of c‐FLIP and Ku70 was analysed using western blot and immunofluorescence. Dissociation of c‐FLIP/Ku70 complex as well as Ku70 translocation were studied by sub‐cellular fractionation and co‐immunoprecipitation. EGCG induced apoptosis in MKN‐45 cells with substantial up‐regulation of P53 and P21, down‐regulation of c‐Myc and Cyclin D1 as well as cell cycle arrest in S and G2/M check points. Moreover, EGCG treatment suppressed the expression of c‐FLIP and Ku70, decreased their interaction while increasing the Ku70 nuclear content. By dissociating the c‐FLIP/Ku70 complex, EGCG could be an alternative component to the conventional HDAC inhibitors in order to induce apoptosis in GC cells. Thus, its combination with other cancer therapy protocols could result in a better therapeutic outcome.

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. A streamlined CRISPR-Cas9-based toolbox that improves the engineering process of Komagataella phaffii yeast by implementing short single-stranded DNA sequences for precise introduction of desired genetic modifications.

Trichosporon asahii is a pathogenic fungus that causes severe, deep-seated fungal infections in neutropenic patients . Elucidating the infection mechanisms of T. asahii based on genetic studies requires a specific gene-targeting system. Here, we established an efficient gene-targeting system in a highly pathogenic T. asahii strain identified using the silkworm infection model. By comparing the pathogenicity of T. asahii clinical isolates in a silkworm infection model, T. asahii MPU129 was identified as a highly pathogenic strain. Using an Agrobacterium tumefaciens -mediated gene transfer system, we obtained a T. asahii MPU129 mutant lacking the ku70 gene, which encodes the Ku70 protein involved in the non-homologous end-joining repair of DNA double-strand breaks. The ku70 gene-deficient mutant showed higher gene-targeting efficiency than the wild-type strain for constructing a mutant lacking the cnb1 gene, which encodes the beta-subunit of calcineurin. The cnb1 gene-deficient mutant showed reduced pathogenicity against silkworms compared with the parental strain. These results suggest that an efficient gene-targeting system in a highly pathogenic T. asahii strain is a useful tool for elucidating the molecular mechanisms of T. asahii infection.

Double‐strand break (DSB), a significant DNA damage brought on by ionizing radiation, acts as an initiating signal in tumor radiotherapy, causing cancer cells death. The two primary pathways for DNA DSB repair in mammalian cells are nonhomologous end joining (NHEJ) and homologous recombination (HR), which cooperate and compete with one another to achieve effective repair. The DSB repair mechanism depends on numerous regulatory variables. DSB recognition and the recruitment of DNA repair components, for instance, depend on the MRE11–RAD50–NBS1 (MRN) complex and the Ku70/80 heterodimer/DNA–PKcs (DNA–PK) complex, whose control is crucial in determining the DSB repair pathway choice and efficiency of HR and NHEJ. In‐depth elucidation on the DSB repair pathway's molecular mechanisms has greatly facilitated for creation of repair proteins or pathways‐specific inhibitors to advance precise cancer therapy and boost the effectiveness of cancer radiotherapy. The architectures, roles, molecular processes, and inhibitors of significant target proteins in the DSB repair pathways are reviewed in this article. The strategy and application in cancer therapy are also discussed based on the advancement of inhibitors targeted DSB damage response and repair proteins.

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.

Gene targeting is a challenge in Yarrowia lipolytica (Y. lipolytica) where non-homologous end-joining (NHEJ) is predominant over homologous recombination (HR). To improve the frequency and efficiency of HR in Y. lipolytica, the ku70 gene responsible for a double stand break (DSB) repair in the NHEJ pathway was disrupted, and the cell cycle was synchronized to the S-phase with hydroxyurea, respectively. Consequently, the HR frequency was over 46% with very short homology regions (50 bp): the pex10 gene was accurately deleted at a frequency of 60% and the β-carotene biosynthetic genes were integrated at the correct locus at an average frequency of 53%. For repeated use, the URA3 marker gene was also excised and deleted at a frequency of 100% by HR between the 100 bp homology regions flanking the URA3 gene. It was shown that appropriate combination of these chemical and biological approaches was very effective to promote HR and construct genetically modified Y. lipolytica strains for biotechnological applications.

The global population constantly breathes particulate matter with an aerodynamic diameter of ≤10 µm (PM10)—a human carcinogen linked to lung cancer. Previous studies have indicated that PM10 causes DNA damage, including double-strand breaks (DSBs). In particular, DSBs are primarily repaired by the non-homologous end joining (NHEJ) pathway, which is essential for maintaining genomic stability; however, the effects of PM10 exposure on this pathway are unknown. To address this, A549 lung epithelial cells were exposed to 10 µg/cm2 of PM10 for 6, 12, and 24 h. We determined that DSBs increased with prolonged exposure, and an increase in the frequency of micronuclei was found. Despite the accumulated DNA damage, no changes in the cell cycle were observed. Reductions in the levels of the Ku80 gene and protein, as well as the Ku70–Ku80 heterodimer—which is essential for initiating NHEJ-mediated repair—were observed. Levels of Artemis (which is responsible for processing DNA damage) remained stable, while levels of the XRCC4 gene and protein (responsible for completing repair) decreased. We conclude that PM10 disrupts two key proteins in the NHEJ pathway, impairing the capacity for DSB repair. This could promote the accumulation of DNA damage and induce genomic instability, contributing to the development of cancer.

Chlamydomonas reinhardtii is a model green microalga that has great industrial potential as a sustainable bio‐factory for recombinant protein and high‐value chemical production. Efficient genome editing tools are required to redesign this organism for synthetic biology applications. CRISPR‐Cas editing technologies have already been adapted for gene knockout, transgene knock‐in, and precise gene editing in C. reinhardtii. However, the efficacy of CRISPR/Cas‐mediated gene knockout (KO) is low, which hampers pathway engineering and functional genomic studies. Here we report that co‐delivery of CRISPR‐Cas gene editing reagents with short double‐stranded non‐homologous oligodeoxynucleotides (dsNHO) increases gene knockout efficacy up to 100‐fold in C. reinhardtii. This phenomenon, referred to as non‐homologous oligonucleotide enhancement (NOE), is heavily affected by the length, structure, and chemical modifications of dsNHO, and is largely mediated by the DNA double‐stranded break sensor KU70/80 (KU) heterodimer in a Cas nuclease‐, locus‐, and strain‐independent manner. Our data suggest that dsNHOs disrupt the cell's double‐stranded break (DSB) sensing pathways, consequently shifting the balance of DNA repair from canonical non‐homologous end joining (c‐NHEJ) towards the more error‐prone, microhomology‐mediated end joining (MMEJ), which could be harnessed as a strategy for improving gene KO efficiency in Chlamydomonas and beyond.

Ku70 is a multifunctional protein with pivotal roles in DNA repair via non-homologous end-joining, V(D)J recombination, telomere maintenance, and neuronal apoptosis control. Nonetheless, its regulatory mechanisms remain elusive. Chicken Ku70 ( GdKu70 ) cDNA has been previously cloned, and DT40 cells expressing it have significantly contributed to critical biological discoveries. GdKu70 features an additional 18 amino acids at its N-terminus compared to mammalian Ku70, the biological significance of which remains uncertain. Here, we show that the 5′ flanking sequence of GdKu70 cDNA is not nearly encoded in the chicken genome. Notably, these 18 amino acids result from fusion events involving the NFE2L1 gene on chromosome 27 and the Ku70 gene on chromosome 1. Through experiments using newly cloned chicken Ku70 cDNA and specific antibodies, we demonstrated that Ku70 localizes within the cell nucleus as a heterodimer with Ku80 and promptly accumulates at DNA damage sites following injury. This suggests that the functions and spatiotemporal regulatory mechanisms of Ku70 in chickens closely resemble those in mammals. The insights and resources acquired will contribute to elucidate the various mechanisms by which Ku functions. Meanwhile, caution is advised when interpreting the previous numerous key studies that relied on GdKu70 cDNA and its expressing cells.

Integration of HIV-1 genomic cDNA results in the formation of single-strand breaks in cellular DNA, which must be repaired for efficient viral replication. Post-integration DNA repair mainly depends on the formation of the HIV-1 integrase complex with the Ku70 protein, which promotes DNA-PK assembly at sites of integration and its activation. Here, we have developed a first-class inhibitor of the integrase-Ku70 complex formation that inhibits HIV-1 replication in cell culture by acting at the stage of post-integration DNA repair. This inhibitor, named s17, does not affect the main cellular function of Ku70, namely its participation in the repair of double-strand DNA breaks through the non-homologous end-joining pathway. Using a molecular dynamics approach, we have constructed a model for the interaction of s17 with Ku70. According to this model, the interaction of two phenyl radicals of s17 with the L76 residue of Ku70 is important for this interaction. The requirement of two phenyl radicals in the structure of s17 for its inhibitory properties was confirmed using a set of s17 derivatives. We propose to stimulate compounds that inhibit post-integration repair by disrupting the integrase binding to Ku70 KuINins.