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Cell Proliferation Aug 2023Renal ischemia-reperfusion injury (IRI) is mainly responsible for acute kidney injury for which there is no effective therapy. Accumulating evidence has indicated the...
Renal ischemia-reperfusion injury (IRI) is mainly responsible for acute kidney injury for which there is no effective therapy. Accumulating evidence has indicated the important role of mitophagy in mitochondrial homeostasis under stress. OGG1 (8-oxoguanine DNA glycosylase) is known for functions in excision repair of nuclear and mitochondrial DNA. However, the role of OGG1 in renal IRI remains unclear. Herein, we identified OGG1, induced during IRI, as a key factor mediating hypoxia-reoxygenation-induced apoptosis in vitro and renal tissue damage in a renal IRI model. We demonstrated that OGG1 expression during IRI negatively regulates mitophagy by suppressing the PINK1/Parkin pathway, thereby aggravating renal ischemic injury. OGG1 knockout and pharmacological inhibition attenuated renal IRI, in part by activating mitophagy. Our results elucidated the damaging role of OGG1 activation in renal IRI, which is associated with the regulatory role of the PINK1/Parkin pathway in mitophagy.
Topics: Humans; Mitophagy; Reperfusion Injury; Ubiquitin-Protein Ligases; DNA Glycosylases; Protein Kinases
PubMed: 36788635
DOI: 10.1111/cpr.13418 -
Bulletin Du Cancer Apr 2021Numerous epigenetic alterations are observed in cancer cells, and dysregulation of mono-ubiquitination of histone H2B (H2Bub1) has often been linked to tumorigenesis.... (Review)
Review
Numerous epigenetic alterations are observed in cancer cells, and dysregulation of mono-ubiquitination of histone H2B (H2Bub1) has often been linked to tumorigenesis. H2Bub1 is a dynamic post-translational histone modification associated with transcriptional elongation and DNA damage response. Histone H2B monoubiquitination occurs in the site of lysine 120, written predominantly by E3 ubiquitin ligases RNF20/RNF40 and deubiquitinated by ubiquitin specific peptidase 22 (USP22). RNF20/40 is often altered in the primary tumors including colorectal cancer, breast cancer, ovarian cancer, prostate cancer, and lung cancer, and the loss of H2Bub1 is usually associated with poor prognosis in tumor patients. The purpose of this review is to summarize the current knowledge of H2Bub1 in transcription, DNA damage response and primary tumors. This review also provides novel options for exploiting the potential therapeutic target H2Bub1 in personalized cancer therapy.
Topics: Carcinoma; DNA Damage; DNA Repair; DNA, Neoplasm; Disease Progression; Gene Expression Regulation, Neoplastic; Histones; Humans; Neoplasm Proteins; Neoplasms; Precision Medicine; Protein Processing, Post-Translational; Transcription Elongation, Genetic; Ubiquitin Thiolesterase; Ubiquitin-Protein Ligases; Ubiquitinated Proteins; Ubiquitination
PubMed: 33685627
DOI: 10.1016/j.bulcan.2020.12.007 -
Journal of Experimental & Clinical... Apr 2022Osteosarcoma (OS) is a malignant bone tumor mostly occurring in children and adolescents, while chemotherapy resistance often develops and the mechanisms involved remain...
BACKGROUND
Osteosarcoma (OS) is a malignant bone tumor mostly occurring in children and adolescents, while chemotherapy resistance often develops and the mechanisms involved remain challenging to be fully investigated.
METHODS
Genome-wide CRISPR screening combined with transcriptomic sequencing were used to identify the critical genes of doxorubicin resistance. Analysis of clinical samples and datasets, and in vitro and in vivo experiments (including CCK-8, apoptosis, western blot, qRT-PCR and mouse models) were applied to confirm the function of these genes. The bioinformatics and IP-MS assays were utilized to further verify the downstream pathway. RGD peptide-directed and exosome-delivered siRNA were developed for the novel therapy strategy.
RESULTS
We identified that E3 ubiquitin-protein ligase Rad18 (Rad18) contributed to doxorubicin-resistance in OS. Further exploration revealed that Rad18 interact with meiotic recombination 11 (MRE11) to promote the formation of the MRE11-RAD50-NBS1 (MRN) complex, facilitating the activation of the homologous recombination (HR) pathway, which ultimately mediated DNA damage tolerance and leaded to a poor prognosis and chemotherapy response in patients with OS. Rad18-knockout effectively restored the chemotherapy response in vitro and in vivo. Also, RGD-exosome loading chemically modified siRad18 combined with doxorubicin, where exosome and chemical modification guaranteed the stability of siRad18 and the RGD peptide provided prominent targetability, had significantly improved antitumor activity of doxorubicin.
CONCLUSIONS
Collectively, our study identifies Rad18 as a driver of OS doxorubicin resistance that promotes the HR pathway and indicates that targeting Rad18 is an effective approach to overcome chemotherapy resistance in OS.
Topics: Adolescent; Animals; Antibiotics, Antineoplastic; Bone Neoplasms; Clustered Regularly Interspaced Short Palindromic Repeats; DNA-Binding Proteins; Doxorubicin; Humans; Mice; Osteosarcoma; Ubiquitin-Protein Ligases
PubMed: 35459258
DOI: 10.1186/s13046-022-02344-y -
Cold Spring Harbor Protocols Nov 2020This protocol describes procedures for cloning blunt-ended DNA fragments into linearized plasmid vectors. To obtain the maximum number of "correct" ligation products...
This protocol describes procedures for cloning blunt-ended DNA fragments into linearized plasmid vectors. To obtain the maximum number of "correct" ligation products when cloning blunt-ended target fragments, the two components of DNA in the ligation reaction must be present at an appropriate ratio. If the molar ratio of plasmid vector to target DNA is too high, then the ligation reaction may generate an undesirable number of circular empty plasmids, both monomeric and polymeric; if too low, the ligation reaction may generate an excess of linear and circular homopolymers and heteropolymers of varying sizes, orientations, and compositions. For this reason, the orientation of the foreign DNA and the number of inserts in each recombinant clone must always be validated by restriction endonuclease mapping or some other means.
Topics: Bacteriophage T4; Buffers; Cloning, Molecular; DNA; DNA Ligases; DNA, Recombinant; Escherichia coli; Genetic Vectors; Plasmids; Viral Proteins
PubMed: 33139501
DOI: 10.1101/pdb.prot101246 -
Journal of Translational Medicine Oct 2022DNA ligases are crucial for DNA repair and cell replication since they catalyze the final steps in which DNA breaks are joined. DNA Ligase III (LIG3) exerts a pivotal...
BACKGROUND
DNA ligases are crucial for DNA repair and cell replication since they catalyze the final steps in which DNA breaks are joined. DNA Ligase III (LIG3) exerts a pivotal role in Alternative-Non-Homologous End Joining Repair (Alt-NHEJ), an error-prone DNA repair pathway often up-regulated in genomically unstable cancer, such as Multiple Myeloma (MM). Based on the three-dimensional (3D) LIG3 structure, we performed a computational screening to identify LIG3-targeting natural compounds as potential candidates to counteract Alt-NHEJ activity in MM.
METHODS
Virtual screening was conducted by interrogating the Phenol Explorer database. Validation of binding to LIG3 recombinant protein was performed by Saturation Transfer Difference (STD)-nuclear magnetic resonance (NMR) experiments. Cell viability was analyzed by Cell Titer-Glo assay; apoptosis was evaluated by flow cytometric analysis following Annexin V-7AAD staining. Alt-NHEJ repair modulation was evaluated using plasmid re-joining assay and Cytoscan HD. DNA Damage Response protein levels were analyzed by Western blot of whole and fractionated protein extracts and immunofluorescence analysis. The mitochondrial DNA (mtDNA) copy number was determined by qPCR. In vivo activity was evaluated in NOD-SCID mice subcutaneously engrafted with MM cells.
RESULTS
Here, we provide evidence that a natural flavonoid Rhamnetin (RHM), selected by a computational approach, counteracts LIG3 activity and killed Alt-NHEJ-dependent MM cells. Indeed, Nuclear Magnetic Resonance (NMR) showed binding of RHM to LIG3 protein and functional experiments revealed that RHM interferes with LIG3-driven nuclear and mitochondrial DNA repair, leading to significant anti-MM activity in vitro and in vivo.
CONCLUSION
Taken together, our findings provide proof of concept that RHM targets LIG3 addiction in MM and may represent therefore a novel promising anti-tumor natural agent to be investigated in an early clinical setting.
Topics: Animals; Mice; Annexin A5; DNA Ligase ATP; DNA Ligases; DNA Repair; DNA, Mitochondrial; Flavonoids; Mice, Inbred NOD; Mice, SCID; Multiple Myeloma; Phenols; Recombinant Proteins
PubMed: 36273153
DOI: 10.1186/s12967-022-03705-z -
Journal of the American Chemical Society Oct 2019Engineered 3D DNA crystals are promising scaffolds for bottom-up construction of three-dimensional, macroscopic devices from the molecular level. Nevertheless, this has...
Engineered 3D DNA crystals are promising scaffolds for bottom-up construction of three-dimensional, macroscopic devices from the molecular level. Nevertheless, this has been hindered by the highly constrained conditions for DNA crystals to be stable. Here we report a method to prepare robust 3D DNA crystals by postassembly ligation to remove this constraint. Specifically, sticky ends at crystal contacts were enzymatically ligated, and the covalent bonds significantly enhanced crystal stability, e.g., being stable at 65 °C. This method also enabled the fabrication of DNA crystals with complex architectures including crystal shell, core-shell, and matryoshka dolls. Furthermore, we have demonstrated the applications of the robust DNA crystals in biocatalysis and protein entrapment. Our study removes one key obstacle for the applications of DNA crystals and offers many new opportunities in DNA nanotechnology.
Topics: Crystallization; DNA; DNA Ligases; Microscopy, Electron, Transmission; Nanotechnology; Nucleic Acid Conformation; Stress, Mechanical; X-Ray Diffraction
PubMed: 31553173
DOI: 10.1021/jacs.9b06613 -
Placenta Mar 2021As key components of DNA repair pathways, DNA ligases catalyze the formation of phosphodiester bonds between DNA single strands, which function as a "glue" to seal the...
INTRODUCTION
As key components of DNA repair pathways, DNA ligases catalyze the formation of phosphodiester bonds between DNA single strands, which function as a "glue" to seal the DNA breaks. DNA ligases play important roles in almost all the normal physiological processes for maintaining the stability of genomic DNA, but their functions in recurrent pregnancy loss (RPL) are still unclear.
METHODS
Immunoblotting was used to determine protein level. DNA damages were examined by comet assay and cell viability was quantified by MTT assay. The cell apoptosis and cell cycle were examined by flow cytometry. The LIG4 mRNA degradation was quantified by qRT-PCR after actinomycin D treatment. The interactions between miRNAs and LIG4 were predicted by TargetScan and confirmed by dual luciferase assay.
RESULTS
LIG1 and LIG4 were downregulated in RPL patients, while γH2AX level was upregulated. Knockdown LIG1 and LIG4 increased DNA damages in trophoblasts, which further induced apoptosis and cell cycle arrest. Serine/arginine-rich splicing factor 1(SRSF1) was reduced in RPL patients and positively correlated with LIG1. Knockdown SRSF1 increased the degradation of LIG1 mRNA which further repressed LIG1 expression. MiR-383 was upregulated in RPL patients and repressed LIG4 expression through interacting with 3'UTR of LIG4 mRNA. The level of miR-383 was found negatively correlated with LIG4 protein level in trophoblasts from RPL patients.
DISCUSSION
LIG1 and LIG4 are downregulated in patients with RPL owing to abnormal RNA degradation and dysregulated miRNA expression. LIG1 and LIG4 downregulation might contribute to the pathophysiological processes of RPL by increasing DNA damages.
Topics: Abortion, Habitual; Adult; DNA Damage; DNA Ligases; Down-Regulation; Female; Humans; Pregnancy; Trophoblasts; Young Adult
PubMed: 33601221
DOI: 10.1016/j.placenta.2021.02.001 -
The FEBS Journal May 2022The chromatin-based DNA damage response pathway is tightly orchestrated by histone post-translational modifications, including histone H2A ubiquitination. Ubiquitination... (Review)
Review
The chromatin-based DNA damage response pathway is tightly orchestrated by histone post-translational modifications, including histone H2A ubiquitination. Ubiquitination plays an integral role in regulating cellular processes including DNA damage signaling and repair. The ubiquitin E3 ligase RNF168 is essential in assembling a cohort of DNA repair proteins at the damaged chromatin via its enzymatic activity. RNF168 ubiquitinates histone H2A(X) at the N terminus and generates a specific docking scaffold for ubiquitin-binding motif-containing proteins. The regulation of RNF168 at damaged chromatin and the mechanistic implication in the recruitment of DNA repair proteins to the damaged sites remain an area of active investigation. Here, we review the function and regulation of RNF168 in the context of ubiquitin-mediated DNA damage signaling and repair. We will also discuss the unanswered questions that require further investigation and how understanding RNF168 targeting specificity could benefit the therapeutic development for cancer treatment.
Topics: Chromatin; DNA Damage; Histones; Humans; Ubiquitin; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 33797206
DOI: 10.1111/febs.15857 -
Molecular Biology Research... Mar 2022Recombinant DNA technology has been playing the key role for a long time since its first beginning. DNA ligases have certainly contributed to the development of cloning...
Recombinant DNA technology has been playing the key role for a long time since its first beginning. DNA ligases have certainly contributed to the development of cloning techniques, as well as molecular study up to now. Despite being a prime cloning tool, DNA ligases still face some shortcomings which lead to their limit of use. Our study provided an improved method that simplified the basic restriction enzyme-based cloning (REC) by eliminating the ligation role, named recombinase-free cloning (RFC). This improved technique was designed with only one PCR reaction, one digestion reaction, and one temperature profile, which takes advantage of endogenous recombinase in host to create the target recombinant vector inside the cell. All purification steps were eliminated for effectively material- and time-saving. Five different clones were generated by RFC. This method showed relatively low efficiency yet successful at a range of 100% in every conducted trial with fragment sizes from 0.5-1.0 kbp. The RFC method could be completed within a day (about 9 hours), without the need of ligase or recombinase or purification steps, which significantly saved DNA components, materials as well as the time required. In conclusion, we expected to provide a more convenient cloning method, as well as enable faster generation of DNA clones, which would be well applied in the less equipped laboratories.
PubMed: 35463820
DOI: 10.22099/mbrc.2021.41923.1685 -
Methods in Enzymology 2020DNA ligases have numerous applications in molecular biology and biotechnology. However, many of these applications require the ligation of blunt-ended DNA termini, which...
DNA ligases have numerous applications in molecular biology and biotechnology. However, many of these applications require the ligation of blunt-ended DNA termini, which is an inefficient activity for existing commercial ligases. To address this limitation, we describe a compartmentalised self-replication protocol that enables enrichment of the most active ligase variants from an arrayed gene library, e.g., for directed evolution. This protocol employs microwell cultures of Escherichia coli cells expressing individual ligase gene variants as both a source of template DNA to generate blunt-ended linear plasmid amplicons, and a source of expressed ligase to circularise its own plasmid amplicon. Transformation of E. coli with the pooled ligation products enables enrichment for clones expressing the most active ligase variants over successive rounds. To facilitate the evaluation of selected ligases, we also describe an in vitro ligation protocol utilising fluorescently labelled, phosphorylated oligonucleotides that are resolved by electrophoresis on a denaturing acrylamide gel to separate the substrate and product bands resulting from blunt-ended, cohesive-ended or nick-sealing ligations.
Topics: DNA Ligases; Escherichia coli; Gene Library; Ligases; Plasmids
PubMed: 32943146
DOI: 10.1016/bs.mie.2020.04.061