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The Journal of Biological Chemistry Jun 2022NAD is a versatile biomolecule acting as a master regulator and substrate in various cellular processes, including redox regulation, metabolism, and various signaling... (Review)
Review
NAD is a versatile biomolecule acting as a master regulator and substrate in various cellular processes, including redox regulation, metabolism, and various signaling pathways. In this article, we concisely and critically review the role of NAD in mechanisms promoting genome maintenance. Numerous NAD-dependent reactions are involved in the preservation of genome stability, the cellular DNA damage response, and other pathways regulating nucleic acid metabolism, such as gene expression and cell proliferation pathways. Of note, NAD serves as a substrate to ADP-ribosyltransferases, sirtuins, and potentially also eukaryotic DNA ligases, all of which regulate various aspects of DNA integrity, damage repair, and gene expression. Finally, we critically analyze recent developments in the field as well as discuss challenges associated with therapeutic actions intended to raise NAD levels.
Topics: ADP Ribose Transferases; DNA; DNA Ligases; Genomic Instability; NAD; Signal Transduction; Sirtuins
PubMed: 35595095
DOI: 10.1016/j.jbc.2022.102037 -
Biochemical and Biophysical Research... Feb 2018Klenow and Klentaq are the large fragment domains of the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus, respectively. Herein, we show that both...
Klenow and Klentaq are the large fragment domains of the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus, respectively. Herein, we show that both polymerases can significantly stimulate complementary intermolecular end-joining ligations by E.coli DNA ligase when the polymerases are present at concentrations lower than that of the DNA substrates. In contrast, high polymerase concentrations relative to the DNA substrates inhibit the intermolecular ligation activity of DNA ligase. Neither polymerase was able to stimulate the DNA ligase from T4 bacteriophage. Additionally, nick-closure by E. coli DNA ligase (but not T4 ligase) is slightly stimulated by both polymerases, but only at about 10% of the magnitude seen for end-joining enhancement. The data represent one of the first observations of direct polymerase-ligase interactions in prokaryotes, and suggest that the polymerases stabilize the associated DNA ends during intermolecular ligation, and that such a complex can be taken advantage of by some, but not all, DNA ligases.
Topics: DNA Damage; DNA End-Joining Repair; DNA Ligases; DNA Polymerase I; DNA Replication; Escherichia coli
PubMed: 29409896
DOI: 10.1016/j.bbrc.2018.01.165 -
Current Medicinal Chemistry 2015About 500 NAD (P)-dependent enzymes in the cell use NAD (P) as a cofactor or a substrate. This family of broadly diversified enzymes is crucial for maintaining... (Review)
Review
About 500 NAD (P)-dependent enzymes in the cell use NAD (P) as a cofactor or a substrate. This family of broadly diversified enzymes is crucial for maintaining homeostasis of all living organisms. The NAD binding domain of these enzymes is conserved and it was believed that NAD mimics would not be of therapeutic value due to lack of selectivity. Consequently, only mycophenolic acid which selectively binds at the cofactor pocket of NAD-dependent IMP-dehydrogenase (IMPDH) has been approved as an immunosuppressant. Recently, it became clear that the NAD (P)-binding domain was structurally much more diversified than anticipated and numerous highly potent and selective inhibitors of NAD (P) dependent enzymes have been reported. It is likely, that as in the case of protein kinases inhibitors, inhibitors of NAD (P)-dependent enzymes would find soon their way to the clinic. In this review, recent developments of selective inhibitors of NAD-dependent human IMPDH, as well as inhibitors of IMPDHs from parasites, and from bacterial sources are reported. Therapies against Cryptosporidium parvum and the development of new antibiotics that are on the horizon will be discussed. New inhibitors of bacterial NAD-ligases, NAD-kinases, NMN-adenylyl transferases, as well as phosphoribosyl transferases are also described. Although none of these compounds has yet to be approved, the progress in revealing and understanding crucial factors that might allow for designing more potent and efficient drug candidates is enormous and highly encouraging.
Topics: Antineoplastic Agents; Bacteria; Binding Sites; Cell Survival; DNA Ligases; Enzyme Inhibitors; Humans; IMP Dehydrogenase; Molecular Dynamics Simulation; NAD; Neoplasms; Phosphotransferases (Alcohol Group Acceptor)
PubMed: 26295463
DOI: 10.2174/0929867322666150821100720 -
BioTechniques Sep 2015The demand for cloned genes has increased incessantly over the past 32 years, but some who need recombinant plasmids struggle to produce them. While the pitfalls of... (Review)
Review
The demand for cloned genes has increased incessantly over the past 32 years, but some who need recombinant plasmids struggle to produce them. While the pitfalls of traditional ligation-dependent cloning are non-trivial, most can be avoided with sufficient effort and attention to detail. Here, the chemical properties of enzymes and reagents used to clone genes into plasmids are reviewed to draw attention to the most pertinent details. In particular, the virtues of agarose gel electrophoresis monitoring, the nature of the interactions between DNA and silica, and challenges associated with thermostable DNA polymerases, restriction endonucleases, and T4 DNA ligase are explored. Common pitfalls associated with Escherichia coli transformation and DNA modifying enzymes are also described. A thorough understanding of established methods is essential for troubleshooting, implementing alternative approaches, and inventing new techniques in response to changes in technology and demand.
Topics: Cloning, Molecular; DNA; DNA Ligases; DNA Restriction Enzymes; Electrophoresis, Agar Gel; Enzymes; Equipment Design; Escherichia coli; Molecular Biology; Polymerase Chain Reaction
PubMed: 26345511
DOI: 10.2144/000114324 -
Nucleic Acids Research Aug 2021The low thermal stability of DNA nanostructures is the major drawback in their practical applications. Most of the DNA nanotubes/tiles and the DNA origami structures...
The low thermal stability of DNA nanostructures is the major drawback in their practical applications. Most of the DNA nanotubes/tiles and the DNA origami structures melt below 60°C due to the presence of discontinuities in the phosphate backbone (i.e., nicks) of the staple strands. In molecular biology, enzymatic ligation is commonly used to seal the nicks in the duplex DNA. However, in DNA nanotechnology, the ligation procedures are neither optimized for the DNA origami nor routinely applied to link the nicks in it. Here, we report a detailed analysis and optimization of the conditions for the enzymatic ligation of the staple strands in four types of 2D square lattice DNA origami. Our results indicated that the ligation takes overnight, efficient at 37°C rather than the usual 16°C or room temperature, and typically requires much higher concentration of T4 DNA ligase. Under the optimized conditions, up to 10 staples ligation with a maximum ligation efficiency of 55% was achieved. Also, the ligation is found to increase the thermal stability of the origami as low as 5°C to as high as 20°C, depending on the structure. Further, our studies indicated that the ligation of the staple strands influences the globular structure/planarity of the DNA origami, and the origami is more compact when the staples are ligated. The globular structure of the native and ligated origami was also found to be altered dynamically and progressively upon ethidium bromide intercalation in a concentration-dependent manner.
Topics: DNA; DNA Ligases; Electrophoresis, Agar Gel; Ethidium; Kinetics; Microscopy, Atomic Force; Nanostructures; Nucleic Acid Conformation; Nucleic Acid Denaturation; Phosphorylation; Temperature; Thermodynamics
PubMed: 34289063
DOI: 10.1093/nar/gkab611 -
Brain : a Journal of Neurology Jun 2021Abnormal gut motility is a feature of several mitochondrial encephalomyopathies, and mutations in genes such as TYMP and POLG, have been linked to these rare diseases....
Abnormal gut motility is a feature of several mitochondrial encephalomyopathies, and mutations in genes such as TYMP and POLG, have been linked to these rare diseases. The human genome encodes three DNA ligases, of which only one, ligase III (LIG3), has a mitochondrial splice variant and is crucial for mitochondrial health. We investigated the effect of reduced LIG3 activity and resulting mitochondrial dysfunction in seven patients from three independent families, who showed the common occurrence of gut dysmotility and neurological manifestations reminiscent of mitochondrial neurogastrointestinal encephalomyopathy. DNA from these patients was subjected to whole exome sequencing. In all patients, compound heterozygous variants in a new disease gene, LIG3, were identified. All variants were predicted to have a damaging effect on the protein. The LIG3 gene encodes the only mitochondrial DNA (mtDNA) ligase and therefore plays a pivotal role in mtDNA repair and replication. In vitro assays in patient-derived cells showed a decrease in LIG3 protein levels and ligase activity. We demonstrated that the LIG3 gene defects affect mtDNA maintenance, leading to mtDNA depletion without the accumulation of multiple deletions as observed in other mitochondrial disorders. This mitochondrial dysfunction is likely to cause the phenotypes observed in these patients. The most prominent and consistent clinical signs were severe gut dysmotility and neurological abnormalities, including leukoencephalopathy, epilepsy, migraine, stroke-like episodes, and neurogenic bladder. A decrease in the number of myenteric neurons, and increased fibrosis and elastin levels were the most prominent changes in the gut. Cytochrome c oxidase (COX) deficient fibres in skeletal muscle were also observed. Disruption of lig3 in zebrafish reproduced the brain alterations and impaired gut transit in vivo. In conclusion, we identified variants in the LIG3 gene that result in a mitochondrial disease characterized by predominant gut dysmotility, encephalopathy, and neuromuscular abnormalities.
Topics: Animals; DNA Ligase ATP; Female; Gastrointestinal Diseases; Gastrointestinal Motility; Humans; Male; Mitochondrial Encephalomyopathies; Mutation; Pedigree; Poly-ADP-Ribose Binding Proteins; Zebrafish
PubMed: 33855352
DOI: 10.1093/brain/awab056 -
Nature Communications Dec 2022During lagging strand synthesis, DNA Ligase 1 (Lig1) cooperates with the sliding clamp PCNA to seal the nicks between Okazaki fragments generated by Pol δ and Flap...
During lagging strand synthesis, DNA Ligase 1 (Lig1) cooperates with the sliding clamp PCNA to seal the nicks between Okazaki fragments generated by Pol δ and Flap endonuclease 1 (FEN1). We present several cryo-EM structures combined with functional assays, showing that human Lig1 recruits PCNA to nicked DNA using two PCNA-interacting motifs (PIPs) located at its disordered N-terminus (PIP) and DNA binding domain (PIP). Once Lig1 and PCNA assemble as two-stack rings encircling DNA, PIP is released from PCNA and only PIP is required for ligation to facilitate the substrate handoff from FEN1. Consistently, we observed that PCNA forms a defined complex with FEN1 and nicked DNA, and it recruits Lig1 to an unoccupied monomer creating a toolbelt that drives the transfer of DNA to Lig1. Collectively, our results provide a structural model on how PCNA regulates FEN1 and Lig1 during Okazaki fragments maturation.
Topics: Humans; DNA Replication; Proliferating Cell Nuclear Antigen; DNA Polymerase III; Ligases; DNA; Flap Endonucleases; DNA Ligase ATP
PubMed: 36539424
DOI: 10.1038/s41467-022-35475-z -
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 -
Analytical Biochemistry Mar 2021The functionalization of 5'-OH group in nucleic acids is of significant value for molecular biology. In the current work we discovered that acid-labile...
The functionalization of 5'-OH group in nucleic acids is of significant value for molecular biology. In the current work we discovered that acid-labile 4,4'-dimethoxytrityl protecting group (DMT) of oligonucleotides (ONs) is stable under PCR conditions and does not interfere with activity of DNA polymerases. So application of 5'-DMT-protected ONs could allow producing both symmetric and asymmetric 5'-DMT-blocked double-stranded DNA (dsDNA) fragments. We demonstrated that the presence of thiol compounds (mercaptoethanol and dithiothreitol) in PCR mixture is undesirable for the stability of DMT-group. DMT-ONs can be successfully used during polymerase chain assembly of synthetic genes. We tested 5'-DMT dsDNA in blunt-end DNA ligation reaction by T4 DNA ligase and found that it could not be ligated with 5'-phosphorylated DNA fragments, namely linearized plasmid vector pJET1.2/blunt. Possible reason for this is steric hindrance created by bulky and rigid DMT-group, that prevents entering enzyme active site. We also demonstrated that 5'-DMT modification of dsDNA does not affect activity of T5 5',3'-exonuclease towards both ssDNA and dsDNA. Further screening of the exonucleases, sensitive to 5'-DMT-modification or search of ways to separate long 5'-DMT-ssDNA and 5'-OH-ssDNA could allow finding application of 5'-DMT-modified oligo- and polynucleotides.
Topics: DNA Ligases; DNA, Single-Stranded; Exodeoxyribonucleases
PubMed: 33508272
DOI: 10.1016/j.ab.2021.114115 -
Methods in Molecular Biology (Clifton,... 2023Endogenous and exogenous genotoxic agents can generate various types of non-ligatable DNA ends at the site of strand break in the mammalian genome. If not repaired, such...
Endogenous and exogenous genotoxic agents can generate various types of non-ligatable DNA ends at the site of strand break in the mammalian genome. If not repaired, such lesions will impede transcription and replication and can lead to various cellular pathologies. Among various "dirty" DNA ends, 3'-phosphate is one of the most abundant lesions generated in the mammalian cells. Polynucleotide kinase 3'-phosphatase (PNKP) is the major DNA end-processing enzyme for resolving 3'-phosphate termini in the mammalian cells, and thus, it is involved in DNA base excision repair (BER), single-strand break repair, and classical nonhomologous end joining (C-NHEJ)-mediated DNA double-strand break (DSB) repair. The 3'-OH ends generated following PNKP-mediated processing of 3'-P are utilized by a DNA polymerase to fill in the gap, and subsequently, the nick is sealed by a DNA ligase to complete the repair process. Here we describe two novel assay systems to detect phosphate release by PNKP's 3'-phosphatase activity and PNKP-mediated in vitro single-strand break repair with minimal repair components (PNKP, DNA polymerase, and DNA ligase) using either purified proteins or cell-free nuclear extracts from mammalian cells/tissues. These assays are highly reproducible and sensitive, and the researchers would be able to detect any significant difference in PNKP's 3'-phosphatase activity as well as PNKP-mediated single-strand break repair activity in diseased mammalian cells/tissues vs normal healthy controls.
Topics: Animals; DNA Repair Enzymes; Polynucleotide 5'-Hydroxyl-Kinase; Radioactivity; DNA Repair; DNA Ligases; DNA-Directed DNA Polymerase; DNA; Phosphates; Phosphoric Monoester Hydrolases; Mammals
PubMed: 37574474
DOI: 10.1007/978-1-0716-3373-1_3