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Cell Cycle (Georgetown, Tex.) Nov 2011DNA ligases are crucial for most DNA transactions, including DNA replication, repair, and recombination. Recently, DNA ligase III (Lig3) has been demonstrated to be... (Review)
Review
DNA ligases are crucial for most DNA transactions, including DNA replication, repair, and recombination. Recently, DNA ligase III (Lig3) has been demonstrated to be crucial for cell survival due to its catalytic function in mitochondria. This review summarizes these recent results and reports on a hitherto unappreciated widespread phylogenetic presence of Lig3 in eukaryotes, including in some organisms before the divergence of metazoa. Analysis of these putative Lig3 homologs suggests that many of them are likely to be found in mitochondria and to be critical for mitochondrial function.
Topics: Conserved Sequence; DNA Ligase ATP; DNA Ligases; Eukaryota; Evolution, Molecular; Mitochondria; Phylogeny; Poly-ADP-Ribose Binding Proteins; Xenopus Proteins
PubMed: 22041657
DOI: 10.4161/cc.10.21.18094 -
Molecules (Basel, Switzerland) Apr 2021Present in all organisms, DNA ligases catalyse the formation of a phosphodiester bond between a 3' hydroxyl and a 5' phosphate, a reaction that is essential for...
Present in all organisms, DNA ligases catalyse the formation of a phosphodiester bond between a 3' hydroxyl and a 5' phosphate, a reaction that is essential for maintaining genome integrity during replication and repair. Eubacterial DNA ligases use NAD as a cofactor and possess low sequence and structural homology relative to eukaryotic DNA ligases which use ATP as a cofactor. These key differences enable specific targeting of bacterial DNA ligases as an antibacterial strategy. In this study, four small molecule accessible sites within functionally important regions of ligase (EC-LigA) were identified using methods. Molecular docking was then used to screen for small molecules predicted to bind to these sites. Eight candidate inhibitors were then screened for inhibitory activity in an ligase assay. Five of these (geneticin, chlorhexidine, glutathione (reduced), imidazolidinyl urea and 2-(aminomethyl)imidazole) showed dose-dependent inhibition of EC-LigA with half maximal inhibitory concentrations (IC) in the micromolar to millimolar range (11-2600 µM). Two (geneticin and chlorhexidine) were predicted to bind to a region of EC-LigA that has not been directly investigated previously, raising the possibility that there may be amino acids within this region that are important for EC-LigA activity or that the function of essential residues proximal to this region are impacted by inhibitor interactions with this region. We anticipate that the identified small molecule binding sites and inhibitors could be pursued as part of an antibacterial strategy targeting bacterial DNA ligases.
Topics: Binding Sites; DNA Ligases; Enzyme Inhibitors; Escherichia coli; Molecular Docking Simulation
PubMed: 33923034
DOI: 10.3390/molecules26092508 -
Cell Structure and Function Feb 1990Two types of DNA ligase, I and II, have been purified approximately 4,000-fold from mouse testes and 500-fold from nuclei of mouse spermatocytes. DNA ligase I and II...
Two types of DNA ligase, I and II, have been purified approximately 4,000-fold from mouse testes and 500-fold from nuclei of mouse spermatocytes. DNA ligase I and II consisted of single polypeptides with molecular weights of 95,000 and 65,000, respectively, according to the estimation by SDS-polyacrylamide gel electrophoresis and the AMP-binding assay. Ligase activities were higher in premeiotic spermatogonia and spermatocytes than those in liver and bone marrow cells. Moreover, DNA ligase II showed rapid increase during meiotic prophase and a decrease in round spermatids. Since this behavior of DNA ligase II is consistent with that of m-rec and DNA polymerase beta, both of which have been shown to be involved in DNA recombination in meiotic cells, DNA ligase II might be an enzyme which works at the final step of meiotic recombination reaction.
Topics: Adenosine Monophosphate; Animals; DNA Ligase ATP; DNA Ligases; Male; Meiosis; Mice; Molecular Weight; Polynucleotide Ligases; Testis
PubMed: 2340590
DOI: 10.1247/csf.15.67 -
Biochemistry and Cell Biology =... Feb 2013DNA double strand breaks (DSBs), induced by ionizing radiation (IR) and endogenous stress including replication failure, are the most cytotoxic form of DNA damage. In... (Review)
Review
DNA double strand breaks (DSBs), induced by ionizing radiation (IR) and endogenous stress including replication failure, are the most cytotoxic form of DNA damage. In human cells, most IR-induced DSBs are repaired by the nonhomologous end joining (NHEJ) pathway. One of the most critical steps in NHEJ is ligation of DNA ends by DNA ligase IV (LIG4), which interacts with, and is stabilized by, the scaffolding protein X-ray cross-complementing gene 4 (XRCC4). XRCC4 also interacts with XRCC4-like factor (XLF, also called Cernunnos); yet, XLF has been one of the least mechanistically understood proteins and precisely how XLF functions in NHEJ has been enigmatic. Here, we examine current combined structural and mutational findings that uncover integrated functions of XRCC4 and XLF and reveal their interactions to form long, helical protein filaments suitable to protect and align DSB ends. XLF-XRCC4 provides a global structural scaffold for ligating DSBs without requiring long DNA ends, thus ensuring accurate and efficient ligation and repair. The assembly of these XRCC4-XLF filaments, providing both DNA end protection and alignment, may commit cells to NHEJ with general biological implications for NHEJ and DSB repair processes and their links to cancer predispositions and interventions.
Topics: Cell Transformation, Neoplastic; DNA; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Ligase ATP; DNA Ligases; DNA Repair; DNA Repair Enzymes; DNA-Binding Proteins; Humans; Models, Molecular; Protein Binding; Radiation, Ionizing
PubMed: 23442139
DOI: 10.1139/bcb-2012-0058 -
The Journal of Biological Chemistry Jul 1990Mammalian DNA ligase I is presumed to act in DNA replication. Rabbit antibodies against the homogeneous enzyme from calf thymus inhibited DNA ligase I activity and...
Mammalian DNA ligase I is presumed to act in DNA replication. Rabbit antibodies against the homogeneous enzyme from calf thymus inhibited DNA ligase I activity and consistently recognized a single polypeptide of 125 kDa when cells from an established bovine kidney cell line (MDBK) were lysed rapidly by a variety of procedures and subjected to immunoblotting analysis. After biosynthetic labeling of MDBK cells with [35S]methionine, immunoprecipitation experiments revealed a polypeptide of 125 kDa that did not appear when purified calf thymus DNA ligase I was used in competition. A 125-kDa polypeptide was adenylated when immunoprecipitated protein from MDBK cells was incubated with [alpha-32P]ATP. Thus, the apparent molecular mass of the initial translation product is identical or nearly so to that of the purified enzyme. The half-life of the protein is 7 h as determined by pulse-chase experiments in asynchronous MDBK cells. Immunocytochemistry and indirect immunofluorescence experiments showed that DNA ligase I is localized to cell nuclei.
Topics: Adenosine Monophosphate; Animals; Blotting, Western; Cattle; Cell Compartmentation; Cell Nucleus; Cells, Cultured; DNA Ligase ATP; DNA Ligases; Fluorescent Antibody Technique; Molecular Weight; Polynucleotide Ligases; Precipitin Tests
PubMed: 2197279
DOI: No ID Found -
The Journal of Biological Chemistry Feb 1994Bovine DNA ligases I and II were adenylylated in the presence of [alpha-32P]ATP and digested with limiting amounts of trypsin or V8 protease. The generation of... (Comparative Study)
Comparative Study
Bovine DNA ligases I and II were adenylylated in the presence of [alpha-32P]ATP and digested with limiting amounts of trypsin or V8 protease. The generation of radioactive peptides of decreasing size was monitored by polyacrylamide gel electrophoresis and autoradiography. Active site peptides obtained by complete proteolytic digestions with trypsin, V8, or Lys-C protease were also compared. The partial digestion products of DNA ligases I and II were entirely different, with no indication of extensive sequence homology. Furthermore, the sequence of the active site region of DNA ligase I is clearly different from that of DNA ligase II. Similar analysis of a third chromatographically distinct mammalian DNA ligase indicated that it is different from DNA ligase I but related to DNA ligase II.
Topics: Adenosine Monophosphate; Adenosine Triphosphate; Amino Acid Sequence; Animals; Binding Sites; Cattle; DNA Ligase ATP; DNA Ligases; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Humans; Isoenzymes; Molecular Sequence Data; Peptide Fragments; Poly-ADP-Ribose Binding Proteins; Xenopus Proteins
PubMed: 8106423
DOI: No ID Found -
International Journal of Radiation... Jul 2013DNA damage can occur as a result of endogenous metabolic reactions and replication stress or from exogenous sources such as radiation therapy and chemotherapy. DNA... (Review)
Review
DNA damage can occur as a result of endogenous metabolic reactions and replication stress or from exogenous sources such as radiation therapy and chemotherapy. DNA double strand breaks are the most cytotoxic form of DNA damage, and defects in their repair can result in genome instability, a hallmark of cancer. The major pathway for the repair of ionizing radiation-induced DSBs in human cells is nonhomologous end joining. Here we review recent advances on the mechanism of nonhomologous end joining, as well as new findings on its component proteins and regulation.
Topics: Antigens, Nuclear; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Ligase ATP; DNA Ligases; DNA Repair Enzymes; DNA-Activated Protein Kinase; DNA-Binding Proteins; Humans; Ku Autoantigen; Nuclear Proteins; Phosphorylation
PubMed: 23433795
DOI: 10.1016/j.ijrobp.2013.01.011 -
PLoS Pathogens Dec 2017Hepadnavirus covalently closed circular (ccc) DNA is the bona fide viral transcription template, which plays a pivotal role in viral infection and persistence. Upon...
Hepadnavirus covalently closed circular (ccc) DNA is the bona fide viral transcription template, which plays a pivotal role in viral infection and persistence. Upon infection, the non-replicative cccDNA is converted from the incoming and de novo synthesized viral genomic relaxed circular (rc) DNA, presumably through employment of the host cell's DNA repair mechanisms in the nucleus. The conversion of rcDNA into cccDNA requires preparation of the extremities at the nick/gap regions of rcDNA for strand ligation. After screening 107 cellular DNA repair genes, we herein report that the cellular DNA ligase (LIG) 1 and 3 play a critical role in cccDNA formation. Ligase inhibitors or functional knock down/out of LIG1/3 significantly reduced cccDNA production in an in vitro cccDNA formation assay, and in cccDNA-producing cells without direct effect on viral core DNA replication. In addition, transcomplementation of LIG1/3 in the corresponding knock-out or knock-down cells was able to restore cccDNA formation. Furthermore, LIG4, a component in non-homologous end joining DNA repair apparatus, was found to be responsible for cccDNA formation from the viral double stranded linear (dsl) DNA, but not rcDNA. In conclusion, we demonstrate that hepadnaviruses utilize the whole spectrum of host DNA ligases for cccDNA formation, which sheds light on a coherent molecular pathway of cccDNA biosynthesis, as well as the development of novel antiviral strategies for treatment of hepatitis B.
Topics: Cell Line; DNA Ligase ATP; DNA Ligases; DNA Repair; DNA, Circular; DNA, Viral; Gene Knockdown Techniques; Gene Knockout Techniques; HEK293 Cells; Hep G2 Cells; Hepadnaviridae; Hepatitis B virus; Hepatocytes; Host-Pathogen Interactions; Humans; Metabolic Networks and Pathways; Poly-ADP-Ribose Binding Proteins
PubMed: 29287110
DOI: 10.1371/journal.ppat.1006784 -
Current Protocols Mar 2023DNA ligases catalyze the joining of breaks in nucleic acid backbones and are essential enzymes for in vivo genome replication and repair across all domains of life....
DNA ligases catalyze the joining of breaks in nucleic acid backbones and are essential enzymes for in vivo genome replication and repair across all domains of life. These enzymes are also critically important to in vitro manipulation of DNA in applications such as cloning, sequencing, and molecular diagnostics. DNA ligases generally catalyze the formation of a phosphodiester bond between an adjacent 5'-phosphate and 3'-hydroxyl in DNA, but they exhibit different substrate structure preferences, sequence-dependent biases in reaction kinetics, and variable tolerance for mismatched base pairs. Information on substrate structure and sequence specificity can inform both biological roles and molecular biology applications of these enzymes. Given the high complexity of DNA sequence space, testing DNA ligase substrate specificity on individual nucleic acid sequences in parallel rapidly becomes impractical when a large sequence space is investigated. Here, we describe methods for investigating DNA ligase sequence bias and mismatch discrimination using Pacific Biosciences Single-Molecule Real-Time (PacBio SMRT) sequencing technology. Through its rolling-circle amplification methodology, SMRT sequencing can give multiple reads of the same insert. This feature permits high-quality top- and bottom-strand consensus sequences to be determined while preserving information on top-bottom strand mismatches that can be obfuscated or lost when using other sequencing methods. Thus, PacBio SMRT sequencing is uniquely suited to measuring substrate bias and enzyme fidelity through multiplexing a diverse set of sequences in a single reaction. The protocols describe substrate synthesis, library preparation, and data analysis methods suitable for measuring fidelity and bias of DNA ligases. The methods are easily adapted to different nucleic acid substrate structures and can be used to characterize many enzymes under a variety of reaction conditions and sequence contexts in a rapid and high-throughput manner. © 2023 New England Biolabs and The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of overhang DNA substrates for ligation Basic Protocol 2: Preparation of ligation fidelity libraries Support Protocol 1: Preparation of ligation libraries for PacBio Sequel II sequencing Support Protocol 2: Loading and sequencing of a prepared library on the Sequel II instrument Basic Protocol 3: Computational processing of ligase fidelity sequencing data.
Topics: DNA Ligases; Substrate Specificity; DNA Ligase ATP; Sequence Analysis, DNA; Technology
PubMed: 36880776
DOI: 10.1002/cpz1.690 -
The Journal of Biological Chemistry Apr 2008Human DNA ligase III contains an N-terminal zinc finger domain that binds to nicks and gaps in DNA. This small domain has been described as a DNA nick sensor, but it is...
Human DNA ligase III contains an N-terminal zinc finger domain that binds to nicks and gaps in DNA. This small domain has been described as a DNA nick sensor, but it is not required for DNA nick joining activity in vitro. In light of new structural information for mammalian ligases, we measured the DNA binding affinity and specificity of each domain of DNA ligase III. These studies identified two separate, independent DNA-binding modules in DNA ligase III that each bind specifically to nicked DNA over intact duplex DNA. One of these modules comprises the zinc finger domain and DNA-binding domain, which function together as a single DNA binding unit. The catalytic core of ligase III is the second DNA nick-binding module. Both binding modules are required for ligation of blunt ended DNA substrates. Although the zinc finger increases the catalytic efficiency of nick ligation, it appears to occupy the same binding site as the DNA ligase III catalytic core. We present a jackknife model for ligase III that posits conformational changes during nick sensing and ligation to extend the versatility of the enzyme.
Topics: Amino Acid Sequence; Biochemistry; Catalytic Domain; Cloning, Molecular; DNA; DNA Ligase ATP; DNA Ligases; Gene Deletion; Humans; Kinetics; Models, Molecular; Molecular Sequence Data; Poly-ADP-Ribose Binding Proteins; Protein Binding; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Xenopus Proteins; Zinc Fingers
PubMed: 18238776
DOI: 10.1074/jbc.M708175200