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Cell Nov 2001Two recent studies illustrate the ability of the Mre11/Rad50 DNA repair complex to bind and connect DNA ends. Specific stimulation of DNA ligase IV-mediated end-joining... (Review)
Review
Two recent studies illustrate the ability of the Mre11/Rad50 DNA repair complex to bind and connect DNA ends. Specific stimulation of DNA ligase IV-mediated end-joining by Mre11 complexes from S. cerevisiae suggests the possibility of a direct role in nonhomologous end-joining in eukaryotic cells.
Topics: DNA; DNA Ligase ATP; DNA Ligases; DNA Repair; DNA-Binding Proteins; Endodeoxyribonucleases; Exodeoxyribonucleases; Fungal Proteins; Humans; Models, Molecular; Protein Structure, Secondary; Protein Structure, Tertiary; Recombination, Genetic; Saccharomyces cerevisiae Proteins
PubMed: 11733056
DOI: 10.1016/s0092-8674(01)00591-8 -
Biochemistry Jul 2010Human DNA ligase III has essential functions in nuclear and mitochondrial DNA replication and repair and contains a PARP-like zinc finger (ZnF) that increases the extent...
Human DNA ligase III has essential functions in nuclear and mitochondrial DNA replication and repair and contains a PARP-like zinc finger (ZnF) that increases the extent of DNA nick joining and intermolecular DNA ligation, yet the bases for ligase III specificity and structural variation among human ligases are not understood. Here combined crystal structure and small-angle X-ray scattering results reveal dynamic switching between two nick-binding components of ligase III: the ZnF-DNA binding domain (DBD) forms a crescent-shaped surface used for DNA end recognition which switches to a ring formed by the nucleotidyl transferase (NTase) and OB-fold (OBD) domains for catalysis. Structural and mutational analyses indicate that high flexibility and distinct DNA binding domain features in ligase III assist both nick sensing and the transition from nick sensing by the ZnF to nick joining by the catalytic core. The collective results support a "jackknife model" in which the ZnF loads ligase III onto nicked DNA and conformational changes deliver DNA into the active site. This work has implications for the biological specificity of DNA ligases and functions of PARP-like zinc fingers.
Topics: Amino Acid Sequence; Catalysis; Crystallography, X-Ray; DNA; DNA Ligase ATP; DNA Ligases; Humans; Models, Molecular; Molecular Sequence Data; Poly(ADP-ribose) Polymerases; Poly-ADP-Ribose Binding Proteins; Protein Conformation; Scattering, Radiation; Xenopus Proteins; Zinc Fingers
PubMed: 20518483
DOI: 10.1021/bi100503w -
Nucleic Acids Research Feb 2014Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of...
Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of RNA-splinted DNA by Chlorella virus DNA ligase (PBCV-1 DNA ligase). PBCV-1 DNA ligase ligated ssDNA splinted by RNA with kcat ≈ 8 x 10(-3) s(-1) and K(M) < 1 nM at 25 °C under conditions where T4 DNA ligase produced only 5'-adenylylated DNA with a 20-fold lower kcat and a K(M) ≈ 300 nM. The rate of ligation increased with addition of Mn(2+), but was strongly inhibited by concentrations of NaCl >100 mM. Abortive adenylylation was suppressed at low ATP concentrations (<100 µM) and pH >8, leading to increased product yields. The ligation reaction was rapid for a broad range of substrate sequences, but was relatively slower for substrates with a 5'-phosphorylated dC or dG residue on the 3' side of the ligation junction. Nevertheless, PBCV-1 DNA ligase ligated all sequences tested with 10-fold less enzyme and 15-fold shorter incubation times than required when using T4 DNA ligase. Furthermore, this ligase was used in a ligation-based detection assay system to show increased sensitivity over T4 DNA ligase in the specific detection of a target mRNA.
Topics: DNA; DNA Ligases; Kinetics; RNA; Viral Proteins
PubMed: 24203707
DOI: 10.1093/nar/gkt1032 -
Nucleic Acids Research 2006DNA ligation is an essential step in DNA replication, repair and recombination. Mammalian cells contain three DNA Ligases that are not interchangeable although they use... (Comparative Study)
Comparative Study
DNA ligation is an essential step in DNA replication, repair and recombination. Mammalian cells contain three DNA Ligases that are not interchangeable although they use the same catalytic reaction mechanism. To compare the recruitment of the three eukaryotic DNA Ligases to repair sites in vivo we introduced DNA lesions in human cells by laser microirradiation. Time lapse microscopy of fluorescently tagged proteins showed that DNA Ligase III accumulated at microirradiated sites before DNA Ligase I, whereas we could detect only a faint accumulation of DNA Ligase IV. Recruitment of DNA Ligase I and III to repair sites was cell cycle independent. Mutational analysis and binding studies revealed that DNA Ligase I was recruited to DNA repair sites by interaction with PCNA while DNA Ligase III was recruited via its BRCT domain mediated interaction with XRCC1. Selective recruitment of specialized DNA Ligases may have evolved to accommodate the particular requirements of different repair pathways and may thus enhance efficiency of DNA repair.
Topics: Animals; Cell Line; DNA Ligase ATP; DNA Ligases; DNA Repair; DNA-Binding Proteins; HeLa Cells; Humans; Kinetics; Mice; Poly-ADP-Ribose Binding Proteins; Proliferating Cell Nuclear Antigen; Protein Transport; X-ray Repair Cross Complementing Protein 1; Xenopus Proteins
PubMed: 16855289
DOI: 10.1093/nar/gkl492 -
Archives of Biochemistry and Biophysics Feb 1986Two DNA ligase activities have been separated, purified, and characterized. The resolution of the two enzymes from crude extracts was initially achieved through Polymin...
Two DNA ligase activities have been separated, purified, and characterized. The resolution of the two enzymes from crude extracts was initially achieved through Polymin P precipitation. The ligation activity precipitating with the nucleic acids on treatment with Polymin P is designated as DNA ligase I, and an activity remaining in the supernatant fraction, as DNA ligase II. DNA ligase I and II are ATP and Mg2+-dependent enzymes with pH optima of 7.8 and 8.0 and isoelectric points of 6.9 and 7.6, respectively. The purified I and II DNA ligase activities have molecular weights of 83,000 and 89,000, respectively. Both activities are inhibited by dATP and inorganic pyrophosphate. However, in the presence of optimum rATP levels, dATP stimulates DNA ligase II activity, whereas DNA ligase I is inhibited under the same conditions. Both activities are DNA specific and ligation follows reaction steps similar to those described for the Escherichia coli DNA ligase.
Topics: Adenosine Triphosphate; DNA Ligases; Deoxyadenine Nucleotides; Diphosphates; Female; Humans; Hydrogen-Ion Concentration; Kinetics; Magnesium; Manganese; Molecular Weight; Nucleic Acid Hybridization; Placenta; Polynucleotide Ligases; Pregnancy; Substrate Specificity; Sulfhydryl Reagents
PubMed: 3004351
DOI: 10.1016/0003-9861(86)90649-1 -
Nucleic Acids Research 2007Agrobacterium tumefaciens encodes a single NAD+-dependent DNA ligase and six putative ATP-dependent ligases. Two of the ligases are homologs of LigD, a bacterial enzyme...
Agrobacterium tumefaciens encodes a single NAD+-dependent DNA ligase and six putative ATP-dependent ligases. Two of the ligases are homologs of LigD, a bacterial enzyme that catalyzes end-healing and end-sealing steps during nonhomologous end joining (NHEJ). Agrobacterium LigD1 and AtuLigD2 are composed of a central ligase domain fused to a C-terminal polymerase-like (POL) domain and an N-terminal 3'-phosphoesterase (PE) module. Both LigD proteins seal DNA nicks, albeit inefficiently. The LigD2 POL domain adds ribonucleotides or deoxyribonucleotides to a DNA primer-template, with rNTPs being the preferred substrates. The LigD1 POL domain has no detectable polymerase activity. The PE domains catalyze metal-dependent phosphodiesterase and phosphomonoesterase reactions at a primer-template with a 3'-terminal diribonucleotide to yield a primer-template with a monoribonucleotide 3'-OH end. The PE domains also have a 3'-phosphatase activity on an all-DNA primer-template that yields a 3'-OH DNA end. Agrobacterium ligases C2 and C3 are composed of a minimal ligase core domain, analogous to Mycobacterium LigC (another NHEJ ligase), and they display feeble nick-sealing activity. Ligation at DNA double-strand breaks in vitro by LigD2, LigC2 and LigC3 is stimulated by bacterial Ku, consistent with their proposed function in NHEJ.
Topics: Agrobacterium tumefaciens; Bacterial Proteins; DNA Breaks, Double-Stranded; DNA Ligase ATP; DNA Ligases; DNA Repair; Protein Structure, Tertiary
PubMed: 17488851
DOI: 10.1093/nar/gkm145 -
Nucleic Acids Research Aug 2015Mammalian DNA ligase III (LigIII) functions in both nuclear and mitochondrial DNA metabolism. In the nucleus, LigIII has functional redundancy with DNA ligase I whereas...
Mammalian DNA ligase III (LigIII) functions in both nuclear and mitochondrial DNA metabolism. In the nucleus, LigIII has functional redundancy with DNA ligase I whereas LigIII is the only mitochondrial DNA ligase and is essential for the survival of cells dependent upon oxidative respiration. The unique LigIII zinc finger (ZnF) domain is not required for catalytic activity but senses DNA strand breaks and stimulates intermolecular ligation of two DNAs by an unknown mechanism. Consistent with this activity, LigIII acts in an alternative pathway of DNA double strand break repair that buttresses canonical non-homologous end joining (NHEJ) and is manifest in NHEJ-defective cancer cells, but how LigIII acts in joining intermolecular DNA ends versus nick ligation is unclear. To investigate how LigIII efficiently joins two DNAs, we developed a real-time, fluorescence-based assay of DNA bridging suitable for high-throughput screening. On a nicked duplex DNA substrate, the results reveal binding competition between the ZnF and the oligonucleotide/oligosaccharide-binding domain, one of three domains constituting the LigIII catalytic core. In contrast, these domains collaborate and are essential for formation of a DNA-bridging intermediate by adenylated LigIII that positions a pair of blunt-ended duplex DNAs for efficient and specific intermolecular ligation.
Topics: DNA; DNA End-Joining Repair; DNA Ligase ATP; DNA Ligases; Humans; Models, Molecular; Poly-ADP-Ribose Binding Proteins; Protein Binding; Protein Structure, Tertiary; Xenopus Proteins; Zinc Fingers
PubMed: 26130724
DOI: 10.1093/nar/gkv652 -
PloS One 2017DNA ligases catalyze the repair of phosphate backbone breaks in DNA, acting with highest activity on breaks in one strand of duplex DNA. Some DNA ligases have also been... (Comparative Study)
Comparative Study
DNA ligases catalyze the repair of phosphate backbone breaks in DNA, acting with highest activity on breaks in one strand of duplex DNA. Some DNA ligases have also been observed to ligate two DNA fragments with short complementary overhangs or blunt-ended termini. In this study, several wild-type DNA ligases (phage T3, T4, and T7 DNA ligases, Paramecium bursaria chlorella virus 1 (PBCV1) DNA ligase, human DNA ligase 3, and Escherichia coli DNA ligase) were tested for their ability to ligate DNA fragments with several difficult to ligate end structures (blunt-ended termini, 3'- and 5'- single base overhangs, and 5'-two base overhangs). This analysis revealed that T4 DNA ligase, the most common enzyme utilized for in vitro ligation, had its greatest activity on blunt- and 2-base overhangs, and poorest on 5'-single base overhangs. Other ligases had different substrate specificity: T3 DNA ligase ligated only blunt ends well; PBCV1 DNA ligase joined 3'-single base overhangs and 2-base overhangs effectively with little blunt or 5'- single base overhang activity; and human ligase 3 had highest activity on blunt ends and 5'-single base overhangs. There is no correlation of activity among ligases on blunt DNA ends with their activity on single base overhangs. In addition, DNA binding domains (Sso7d, hLig3 zinc finger, and T4 DNA ligase N-terminal domain) were fused to PBCV1 DNA ligase to explore whether modified binding to DNA would lead to greater activity on these difficult to ligate substrates. These engineered ligases showed both an increased binding affinity for DNA and increased activity, but did not alter the relative substrate preferences of PBCV1 DNA ligase, indicating active site structure plays a role in determining substrate preference.
Topics: DNA Breaks, Double-Stranded; DNA Ligases; Electrophoresis, Capillary; Humans
PubMed: 29284038
DOI: 10.1371/journal.pone.0190062 -
Mutation Research. Reviews in Mutation... Jul 2017Genetic variants have been reported to cause several genetic diseases. Various genotyping assays have been developed for diagnostic and screening purposes but with... (Review)
Review
Genetic variants have been reported to cause several genetic diseases. Various genotyping assays have been developed for diagnostic and screening purposes but with certain limitations in sensitivity, specificity, cost effectiveness and/or time savings. Since the discovery of ligase chain reaction (LCR) in the late nineties, it became one of the most favored platforms for detecting these variants and also for genotyping low abundant contaminants. Recent and powerful modifications with the integration of various detection strategies such as electrochemical and magnetic biosensors, nanoparticles (NPs), quantum dots, quartz crystal and leaky surface acoustic surface biosensors, DNAzyme, rolling circle amplification (RCA), strand displacement amplification (SDA), surface enhanced raman scattering (SERS), chemiluminescence and fluorescence resonance energy transfer have been introduced to both LCR and ligation based amplifications to enable high-throughput and inexpensive multiplex genotyping with improved robustness, simplicity, sensitivity and specificity. In this article, classical and up to date modifications in LCR and ligation based amplifications are critically evaluated and compared with emphasis on points of strength and weakness, sensitivity, cost, running time, equipment needed, applications and multiplexing potential. Versatile genotyping applications such as genetic diseases detection, bacterial and viral pathogens detection are also detailed. Ligation based gold NPs biosensor, ligation based RCA and ligation mediated SDA assays enhanced detection limit tremendously with a discrimination power approaching 1.5aM, 2aM and 0.1fM respectively. MLPA (multiplexed ligation dependent probe amplification) and SNPlex assays have been commercialized for multiplex detection of at least 48 SNPs at a time. MOL-PCR (multiplex oligonucleotide ligation) has high-throughput capability with multiplex detection of 50 SNPs/well in a 96 well plate. Ligase detection reaction (LDR) is one of the most widely used LCR versions that have been successfully integrated with several detection strategies with improved sensitivity down to 0.4fM.
Topics: Biosensing Techniques; DNA Ligases; Genome, Human; Genotyping Techniques; Gold; Humans; Ligase Chain Reaction; Metal Nanoparticles; Polymerase Chain Reaction; Polymorphism, Single Nucleotide
PubMed: 28927538
DOI: 10.1016/j.mrrev.2017.05.001 -
The FEBS Journal Nov 2008DNA ligases are the enzymes essential for DNA replication, repair and recombination in all organisms. The bacterial DNA ligases involved in DNA replication require...
DNA ligases are the enzymes essential for DNA replication, repair and recombination in all organisms. The bacterial DNA ligases involved in DNA replication require NAD(+) for activity, but eukaryotic and viral DNA ligases require ATP. Because of their essential nature, unique structures and widespread existence in nature, bacterial DNA ligases represent a class of valuable targets for identifying novel and selective antibacterial agents. In this study, we cloned and expressed the ligA gene from Streptococcus pneumoniae, and characterized this ligA-encoded NAD(+)-dependent DNA ligase. We then screened small molecule chemical libraries using a biochemical assay and identified a new small molecule with a structure of 2,4-diamino-7-dimethylamino-pyrimido[4,5-d]pyrimidine. We show that this small molecule is a specific inhibitor of bacterial NAD(+)-dependent DNA ligases. Biochemical studies show that this molecule inhibits NAD(+)-dependent DNA ligases, but not ATP-dependent enzymes. The molecule inhibits NAD(+)-dependent DNA ligases competitively with respect to NAD(+) and specifically inhibits enzyme adenylation, but not DNA adenylation or ligation. Labeling studies establish that this molecule inhibits the incorporation of thymidine into DNA and that overexpression of DNA ligase in the cell abolishes this inhibition. Finally, microbiological studies show that this molecule exhibits a broad spectrum of antibacterial activity. Together, this study shows that this small molecule inhibitor identified is specific to bacterial NAD(+)-dependent DNA ligases, exhibits a broad spectrum of antibacterial activities, and has the potential to be developed into an antibacterial agent.
Topics: Anti-Bacterial Agents; Bacterial Proteins; Base Sequence; DNA Ligases; Enzyme Inhibitors; Humans; Kinetics; Pyrimidines; Small Molecule Libraries; Streptococcus pneumoniae
PubMed: 18795946
DOI: 10.1111/j.1742-4658.2008.06652.x