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Discovery and Optimization of NAD+-Dependent DNA Ligase Inhibitors as Novel Antibacterial Compounds.Current Pharmaceutical Design 2017With the serious problem of multiple drug resistance to antibiotics among pathogenic bacteria spreading across the world over the past 30 years, it is crucial to search... (Review)
Review
With the serious problem of multiple drug resistance to antibiotics among pathogenic bacteria spreading across the world over the past 30 years, it is crucial to search for novel inhibitors with distinct modes of action from diverse chemical classes. NAD+-dependent DNA ligases (LigAs) are essential enzymes in bacteria, vital for DNA replication and repair. Additionally, LigAs exclusively exist in eubacteria and some viruses and are not found in humans. Those enzymes have therefore been identified as attractive antibacterial drug targets. In this review we explore the discovered inhibitors of LigA through high-throughput screening or virtual screening respectively and their further structure optimization.
Topics: Animals; Anti-Bacterial Agents; DNA Ligases; Dose-Response Relationship, Drug; Drug Discovery; Drug Evaluation, Preclinical; Enzyme Inhibitors; High-Throughput Screening Assays; Humans; Molecular Structure; NAD; Structure-Activity Relationship
PubMed: 27784238
DOI: 10.2174/1381612822666161025145639 -
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 -
Structure (London, England : 1993) Mar 2022DNA ligases act in the final step of many DNA repair pathways and are commonly regulated by the DNA sliding clamp proliferating cell nuclear antigen (PCNA), but there...
DNA ligases act in the final step of many DNA repair pathways and are commonly regulated by the DNA sliding clamp proliferating cell nuclear antigen (PCNA), but there are limited insights into the physical basis for this regulation. Here, we use single-particle cryoelectron microscopy (cryo-EM) to analyze an archaeal DNA ligase and heterotrimeric PCNA in complex with a single-strand DNA break. The cryo-EM structures highlight a continuous DNA-binding surface formed between DNA ligase and PCNA that supports the distorted conformation of the DNA break undergoing repair and contributes to PCNA stimulation of DNA ligation. DNA ligase is conformationally flexible within the complex, with its domains fully ordered only when encircling the repaired DNA to form a stacked ring structure with PCNA. The structures highlight DNA ligase structural transitions while docked on PCNA, changes in DNA conformation during ligation, and the potential for DNA ligase domains to regulate PCNA accessibility to other repair factors.
Topics: Cryoelectron Microscopy; DNA; DNA Ligase ATP; DNA Ligases; DNA Replication; Nucleic Acid Conformation; Proliferating Cell Nuclear Antigen; Protein Binding
PubMed: 34838188
DOI: 10.1016/j.str.2021.11.002 -
Structure (London, England : 1993) Mar 2022In this issue of Structure, Sverzhinsky et al. (2022) report structures of archaeal DNA ligase bound to the proliferating cell nuclear antigen (PCNA) sliding clamp and...
In this issue of Structure, Sverzhinsky et al. (2022) report structures of archaeal DNA ligase bound to the proliferating cell nuclear antigen (PCNA) sliding clamp and a nicked DNA substrate. The structures provide snapshots of ligation intermediates, which reveal a dynamic nature of the complex and explain how PCNA stimulates the DNA ligase activity.
Topics: Cryoelectron Microscopy; DNA; DNA Ligase ATP; DNA Ligases; Proliferating Cell Nuclear Antigen; Protein Binding
PubMed: 35245433
DOI: 10.1016/j.str.2022.02.008 -
Nature Communications Mar 2024This study establishes the physiological role of Fused in Sarcoma (FUS) in mitochondrial DNA (mtDNA) repair and highlights its implications to the pathogenesis of...
This study establishes the physiological role of Fused in Sarcoma (FUS) in mitochondrial DNA (mtDNA) repair and highlights its implications to the pathogenesis of FUS-associated neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Endogenous FUS interacts with and recruits mtDNA Ligase IIIα (mtLig3) to DNA damage sites within mitochondria, a relationship essential for maintaining mtDNA repair and integrity in healthy cells. Using ALS patient-derived FUS mutant cell lines, a transgenic mouse model, and human autopsy samples, we discovered that compromised FUS functionality hinders mtLig3's repair role, resulting in increased mtDNA damage and mutations. These alterations cause various manifestations of mitochondrial dysfunction, particularly under stress conditions relevant to disease pathology. Importantly, rectifying FUS mutations in patient-derived induced pluripotent cells (iPSCs) preserves mtDNA integrity. Similarly, targeted introduction of human DNA Ligase 1 restores repair mechanisms and mitochondrial activity in FUS mutant cells, suggesting a potential therapeutic approach. Our findings unveil FUS's critical role in mitochondrial health and mtDNA repair, offering valuable insights into the mechanisms underlying mitochondrial dysfunction in FUS-associated motor neuron disease.
Topics: Animals; Humans; Mice; Amyotrophic Lateral Sclerosis; DNA, Mitochondrial; Ligases; Mice, Transgenic; Mitochondrial Diseases; Motor Neuron Disease; Mutation; RNA-Binding Protein FUS; DNA Ligase ATP
PubMed: 38461154
DOI: 10.1038/s41467-024-45978-6 -
Methods in Molecular Biology (Clifton,... 2018In past decades, DNA engineering protocols have led to the rapid development of synthetic biology. To engineer the natural proteins, many directed evolution methods...
In past decades, DNA engineering protocols have led to the rapid development of synthetic biology. To engineer the natural proteins, many directed evolution methods based on molecular biology have been presented for generating genetic diversity or obtaining specific properties. Here, we provide a simple (PCR operation), efficient (larger amount of products), and powerful (multiple point mutations, deletions, insertions, and combinatorial multipoint mutagenesis) RECODE method, which is capable of reediting the target DNA flexibly to restructure regulatory regions and remodel enzymes by using the combined function of the thermostable DNA polymerase and DNA ligase in one pot. RECODE is expected to be an applicable choice to create diverse mutant libraries for rapid evolution and optimization of enzymes and synthetic pathways.
Topics: DNA; DNA Ligases; DNA-Directed DNA Polymerase; Directed Molecular Evolution; Gene Library; Genetic Variation; Mutagenesis; Polymerase Chain Reaction; Synthetic Biology
PubMed: 29754230
DOI: 10.1007/978-1-4939-7795-6_11 -
PloS One 2023DNA Ligase IV is responsible for the repair of DNA double-strand breaks (DSB), including DSBs that are generated during V(D)J recombination. Like other DNA ligases,...
DNA Ligase IV is responsible for the repair of DNA double-strand breaks (DSB), including DSBs that are generated during V(D)J recombination. Like other DNA ligases, Ligase IV contains a catalytic core with three subdomains-the DNA binding (DBD), the nucleotidyltransferase (NTD), and the oligonucleotide/oligosaccharide-fold subdomain (OBD). Ligase IV also has a unique C-terminal region that includes two BRCT domains, a nuclear localization signal sequence and a stretch of amino acid that participate in its interaction with XRCC4. Out of the three mammalian ligases, Ligase IV is the only ligase that participates in and is required for V(D)J recombination. Identification of the minimal domains within DNA Ligase IV that contribute to V(D)J recombination has remained unresolved. The interaction of the Ligase IV DNA binding domain with Artemis, and the interaction of its C-terminal region with XRCC4, suggest that both of these regions that also interact with the Ku70/80 heterodimer are important and might be sufficient for mediating participation of DNA Ligase IV in V(D)J recombination. This hypothesis was investigated by generating chimeric ligase proteins by swapping domains, and testing their ability to rescue V(D)J recombination in Ligase IV-deficient cells. We demonstrate that a fusion protein containing Ligase I NTD and OBDs flanked by DNA Ligase IV DBD and C-terminal region is sufficient to support V(D)J recombination. This chimeric protein, which we named Ligase 37, complemented formation of coding and signal joints. Coding joints generated with Ligase 37 were shorter than those observed with wild type DNA Ligase IV. The shorter length was due to increased nucleotide deletions and decreased nucleotide insertions. Additionally, overexpression of Ligase 37 in a mouse pro-B cell line supported a shift towards shorter coding joints. Our findings demonstrate that the ability of DNA Ligase IV to participate in V(D)J recombination is in large part mediated by its DBD and C-terminal region.
Topics: Animals; Mice; DNA Ligase ATP; V(D)J Recombination; DNA Ligases; Nucleotides; DNA; Mammals
PubMed: 36827388
DOI: 10.1371/journal.pone.0282236