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At least 19 recordsLinked to original sources
PubMed1978

Simultaneous purification and some properties of aspartate: tRNA ligase and seven other amino-acid:tRNA ligases from Escherichia coli.

A procedure is described for the purification of the aspartate:tRNA ligase from Escherichia coli to a stage where it was homogeneous by polyacrylamide gel electrophoresis. From the same batch of E. coli the lysine, phenylalanine and serine ligases were obtained in an apparently homogeneous form while the alanine, glutamine, leucine and valine enzymes had a purity varying from 20% to 80%. Aspartate: tRNA ligase, which has not been obtained in a highly purified form before, has been characterized in terms of its molecular parameters.

Aspartic Acid↗
PubMed1977

Characterization of liver cholic acid coenzyme A ligase activity. Evidence that separate microsomal enzymes are responsible for cholic acid and fatty acid activation.

Investigations on the cholic acid CoA ligase activity of rat liver microsomes were made possible by the development of a rapid, sensitive radiochemical assay based on the conversion of [3H]choloyl-CoA. More than 70% of the rat liver cholic acid CoA ligase activity was associated with the microsomal subcellular fraction. The dependencies of cholic acid CoA ligase activity on pH, ATP, CoA, Triton WR-1339, acetone, ethanol, magnesium, and salts were investigated. The hypothesis that the long chain fatty acid CoA ligase activity and the cholic acid CoA ligase activity are catalyzed by a single microsomal enzyme was investigated. The ATP, CoA, and cholic (palmitic) acid kinetics neither supported nor negated the hypothesis. Cholic acid was not an inhibitor of the fatty acid CoA ligase and palmitic acid was not a competitive inhibitor of the cholic acid CoA ligase. The cholic acid CoA ligase activity utilized dATP as a substrate more effectively than did the fatty acid CoA ligase activity. The cholic acid and fatty acid CoA ligase activities appeared to have different pH dependencies, differed in thermolability at 41 degrees, and were differentially inactivated by phospholipase C. Moreover, fatty acid CoA ligase activity was present in microsomal fractions from all rat organs tested while cholic acid CoA ligase activity was detected only in liver microsomes. The data suggest that separate microsomal enzymes are responsible for the cholic acid and the fatty acid CoA ligase activities in liver.

Adenosine Triphosphate↗
PubMed1979

RNA ligase reaction products in plasmolyzed Escherichia coli cells infected by T4 bacteriophage.

Searching for a physiological role of T4 RNA ligase [polyribonucleotide synthetase (ATP); poly(ribonucleotide):poly(ribonucleotide) ligase (AMP-forming), EC 6.5.1.3] activity, we developed an acellular system of plasmolyzed Escherichia coli cells infected by T4 bacteriophage. Upon incubation of this system with [gamma-32P]ATP, 32P was transferred into a large number of polyribonucleotides, mostly up to 300-400 residues long. The bulk of 32P in the product polyribonucleotides was found in 5'-terminal phosphate groups, suggesting that they originated by a phosphorylation reaction catalyzed by the endogenous polynucleotide kinase (EC 2.7.1.78). Indeed, these products were not seen in an acellular system from uninfected cells, and their amount and complexity increased with the progress of infection. Analysis of the 32P-labeled polyribonucleotide products by gel electrophoresis, either before or after digestion with alkaline phosphatase (EC 3.1.3.1), revealed that a small fraction of the 32P resided in phosphodiester bonds of several tRNA-sized chains. This specific 32P transfer from [gamma-32P]ATP into phosphodiester bonds was apparently catalyzed by successive polynucleotide kinase and RNA ligase reactions. The possible relationship of the 32P transfer to RNA ligase was investigated next by using a system from cells infected with T4 am M69 (an amber mutant deficient in RNA ligase). Transfer of 32P from [gamma-32P]ATP into phosphodiester bonds was not detected in the am M69 system. However, addition of purified RNA ligase to the am M69 system restored the specific 32P transfer. A system from cells infected with T4 psu-b delta 33 (a deletion mutant lacking the entire tRNA region) sustained the specific 32P transfer into tRNA-sized products, indicating that they were not derived from transcripts of T4 tRNA genes. These data may reflect a role of RNA ligase in posttranscriptional conversion of presumably host polyribonucleotides into novel tRNA species during T4 infection.

Adenosine Triphosphate↗
PubMed1979

Functional interactions beween the DNA ligase of Escherichia coli and components of the DNA metabolic apparatus of T4 bacteriophage.

T4 phage completely defective in both gene 30 (DNA ligase) and the rII gene (function unknown) require at least normal levels of host-derived DNA ligase (E. coli lig gene) for growth. Viable E. coli mutant strains that harbor less than 5% of the wild-type level of bacterial ligase do not support growth of T4 doubly defective in genes 30 and rII (T4 30- rII- mutants). We describe here two classes of secondary phage mutations that permit the growth of T4 30- rII- phage on ligase-defective hosts. One class mapped in T4 gene su30 (Krylov 1972) and improved T4 30- rII- phage growth on all E. coli strains, but to varying degrees that depended on levels of residual host ligase. Another class mapped in T4 gene 32 (helix-destabilizing protein) and improved growth specifically on a host carrying the lig2 mutation, but not on a host carrying another lig- lesion (lig4). Two conclusions are drawn from the work: (1) the role of DNA ligase in essential DNA metabolic processes in T4-infected E. coli is catalytic rather than stoichiometric, and (2) the E. coli DNA ligase is capable of specific functional interactions with components of the T4 DNA replication and/or repair apparatus.

Coliphages↗
PubMed2026

Post-translational chemical modification of E3 ligase for efficient target protein degradation.

Targeted protein degradation (TPD) has emerged as a powerful therapeutic strategy, with proteolysis-targeting chimeras (PROTACs) leading efforts to address previously undruggable targets. However, PROTACs face challenges such as low bioavailability and poor pharmacokinetic properties which limit their biological applications. Here, we report a strategy termed post-translational chemical modification targeting chimera (PTcM-TAC), which integrates ligand-directed chemistry into the PROTAC framework to achieve sustained target protein degradation through covalent modification of E3 ligases. PTcM-TAC incorporates an electrophilic dibromophenyl benzoate warhead into the linker connecting the E3 ligase ligand and the protein-of-interest (POI) ligand, enabling selective transfer of the POI ligand onto the recruited E3 ligase while releasing the E3-binding moiety. Mechanistic studies, including LC-MS/MS peptide mapping, pull-down assays, and structural modeling, demonstrated site-selective modification of CRBN by the PTcM-TAC. The resulting ligand-labeled E3 ligase enables sustained pseudo-catalytic target recognition through a simplified binary interaction, thereby maintaining degradation activity even after compound washout. Furthermore, we successfully applied the PTcM-TAC strategy to another representative E3 ligase, von Hippel-Lindau (VHL), which exhibited substantially sustained degradation activity compared with conventional PROTACs. To our knowledge, PTcM-TAC represents the first ligand-directed chemical strategy that converts transient PROTAC-mediated ternary complex formation into binary target recognition via post-translational chemical modification of an E3 ligase. We believe that PTcM-TAC could provide a platform for next-generation targeted protein degraders to overcome the current limitation of PROTAC approach.

Ubiquitin-Protein Ligases↗
PubMed1976

Role of deoxyribonucleic acid polymerases and deoxyribonucleic acid ligase in x-ray-induced repair synthesis in toluene-treated Escherichia coli K-12.

Toluene-treated Escherichia coli mutants have been used to study the roles of deoxyribonucleic acid (DNA) polymerases I, II, and III, and of DNA ligase in repair synthesis and strand rejoining following X-irradiation. In cells possessing all three DNA polymerases, both a greater amount of repair synthesis ("exaggerated" repair synthesis) and failure of ligation are observed when DNA ligase activity is inhibited. In a mutant lacking the polymerizing activity of DNA polymerase I, exaggerated repair synthesis is not observed, and strand rejoining does not occur even if DNA ligase is fully activated. In a mutant possessing the polymerizing activity of DNA polymerase I but lacking its 5'leads to 3' exonuclease activity, exaggerated repair synthesis is minimal. After irradiation, DNA polymerases II and III are capable of carrying out an adenosine 5'-triphosphate-dependent repair synthesis,but rejoining of strand breaks does not occur and exaggerated synthesis is not seen whether DNA ligase is active or not. These results suggest that DNA polymerase I and DNA ligase act together to limit repair synthesis after X irradiation and that both are necessary in toluene-treated cells for strand rejoining. DNA polymerases II and III apparently cannot complete chain elongation and gap filling, and therefore repair carried out by these enzymes does not respond to ligase action.

Adenosine Triphosphate↗
PubMed1977

Bacteriophage T4 RNA ligase: preparation of a physically homogeneous, nuclease-free enzyme from hyperproducing infected cells.

Infection of Escherichia coli by a bacteriophage T4 regA, gene 44 double mutant leads to about a 7-fold increase in the amount of RNA ligase obtained after infection by wild-type phage. Using cells infected by the double mutant, RNA ligase was purified to homogeneity with a 20% yield. Unlike previous preparations of this enzyme, the ligase is free of contaminating nuclease and is therefore suitable for intermolecular ligation of DNA substrates. In the course of these studies it was discovered that adenylalation of the enzyme--a step in the reaction pathway--markedly decreased the electrophoretic mobility of RNA ligase through polyacrylamide gels containing sodium dodecyl sulfate. This behavior allows identification of RNA ligase among a mixture of proteins and was used to demonstrate that virtually all of the purified protein is enzymatically active.

Adenosine Monophosphate↗
PubMed1975

Properties of a DNA-adenylate complex formed in the reaction between mammalian DNA ligase I and DNA containing single-strand breaks.

The major DNA ligase from calf thymus (mammalian DNA ligase I) forms a covalent enzyme-AMP complex on incubation with ATP [Söderhäll & Lindahl, J. Biol. Chem. 248, 672-675, (1973)]. The reaction of this complex with DNA has now been studied. When the ligase-adenylate complex is incubated at 0 degrees C for short time periods with DNA containing single-strand breaks, a DNA-AMP complex can be isolated from the reaction mixture by isopycnic centrifugation in CsCl. Incubation at pH 6.5 increased the amount of DNA-AMP complex that could be isolated 10-20-fold relative to that obtained at pH 7.4. Under the same conditions, incubation of the ligase-AMP complex with DNA free from single-strand breaks did not lead to detectable DNA-AMP formation. The DNA-AMP complex was resistant to treatment with dilute acid and alkali indicating the presence of a covalent linkage. Further, this complex was sensitive to DNase but resistant to pronase and RNase. Free AMP was released on further incubation of the isolated DNA-AMP complex with thymus DNA ligase I and Mg2+, suggesting that the complex is a reaction intermediate. Degradation of the DNA-AMP complex with several reagent enzymes indicated that the AMP residues were bound at the 5' ends of the single-strand breaks in DNA by pyrophosphate bonds.

Adenosine Monophosphate↗
PubMed2026

Virome-wide ubiquitin ligase discovery reveals diverse mechanisms of immune evasion.

Viruses are intracellular parasites that reprogram the host proteome to promote replication and evade immune recognition. We applied a virome-wide library of ~10,000 open reading frames to discover viral ubiquitin ligases, mapping their mechanisms of degradation and host substrates using targeted CRISPR screens and proteomics. These viral effectors could be classified as canonical ligases that mimic host E3s, hijackers that redirect host E3s, and noncanonical ligases that rewire cullin-RING ligase machinery. These diverse strategies of virus-mediated degradation converged on immune-related substrates, including JAK1 and CUL1β-TrCP, underscoring immune evasion as a major driver of viral ubiquitin ligase evolution. Our findings elucidate viral strategies for exploiting the ubiquitin-proteasome system with potential for therapeutic targeting.

Humans↗
PubMed1976

Actions of bleomycin on DNA ligase and polymerases.

Bleomycin inhibited the ligase, which was partially purified from rat ascites hepatoma, AH-130, even at a concentration as low as 0.01-1 mug/ml. The DNA degraded by bleomycin was not repaired by ligase. Therefore, it was suggested that bleomycin at higher concentration produced strand scission of DNA, which could not be repaired by the ligase, and at lower concentration inhibited the ligase reaction presumably by binding to DNA strand or to ligase. Also, the specificity of inhibition by bleomycin on the DNA polymerase of oncogenic RNA virus was tested, comparing with the four kinds of DNA polymerases extracted from the spleen of mice infected with Friend virus. Three kinds of DNA polymerases from spleen were not inhibited by bleomycin, but the fourth enzyme, which was induced in the spleen by virus infection, was inhibited by the antibiotic, when poly-d(AT) and poly-dG with dC were used as template.

Animals↗
PubMed1976

Properties of DNA ligase from uninfected and virus-infected HeLa cells.

HeLa cells contain a high M.W. form of DNA ligase which can be completely converted to a low M.W. form. Stokes radius, frictional ratio, sedimentation coefficient, molecular weight, pH dependence, and heat inactivation rate of the two forms have been studied. The major properties of the two forms of DNA ligase in HeLa cells (in particular molecular weights and pH dependence) resemble those of the "dimer" and "monomer" structures described in cultured human cells (Pedrali, G., Spadari, S., Ciarrocchi, G., Pedrini, M., Falaschi, A. (1973) Eur. J. Biochem., 39 343) . In synchronized HeLa cells, the DNA ligase shows a two fold increase during S phase and parallels the increase in the DNA synthesis rate. DNA ligase increases in parallel with viral DNA synthesis after infection of HeLa cells with vaccinia and Herpes virus but its cofactor requirements and physical properties (including the dimer leads to monomer conversion) are unchanged, suggesting that the newly formed ligase is not virus-coded.

HeLa Cells↗
PubMed1977

Bacteriophage T4 RNA ligase is gene 63 product, the protein that promotes tail fiber attachment to the baseplate.

RNA ligase and tail fiber attachment activities, normally induced following bacteriophage T4 infection of Escherichia coli, are not induced when gene 63 amber mutants of T4 infect nonpermissive host cells. Both activities are induced when these mutants infect permissive hosts, or when revertants of these mutants infect nonpermissive hosts. When one of these mutants infects a host that carries supF, both activities are more than normally heat labile. RNA ligase, purified to homogeneity, promotes the tail fiber attachment reaction in vitro with a specific activity similar to that of the most highly purified preparations of gene 63 product isolated on the basis of tail fiber attachment activity. We conclude that T4 RNA ligase is gene 63 product. The RNA ligase and tail fiber attachment reactions differ in requirements and in response to some inhibitors, suggesting that the two activities of the gene 63 product may be mechanistically unrelated.

Coliphages↗
PubMed1979

Evidence that a single DNA ligase is involved in replication and recombination in yeast.

The possible existence in yeast of different nuclear DNA ligase enzymes led us to ask whether induced recombination (gene conversion) involves the same ligase as that involved in DNA replication. The conditional cdc9 mutant is known to be defective, under restrictive conditions, in the rejoining of Okazaki fragments. We show here that under the same conditions, x-ray-induced convertants within the cdc9 locus are produced with kinetics indicating that most, if not all, of the conversion events require the participation of the cdc9-controlled ligase. Thus, the same DNA ligase is involved in DNA replication and in induced gene conversion.

DNA Ligases↗
PubMed2025

Rice E3 ligase OsRFPH2-16 acts as a negative regulator to mediate the degradation of OsPIP1;1 under salt stress.

Soil salinity has a significant negative effect on rice productivity. We characterized the Oryza sativa RING Finger Protein H2-type-16 gene (OsRFPH2-16), which plays a negative role in response to salinity. The transcript levels of OsRFPH2-16 decreased under saline conditions. OsRFPH2-16 was expressed in the ER and tonoplasts of rice protoplasts. In addition, OsRFPH2-16 exhibited E3 ligase activity in an in vitro ubiquitination assay, whereas the mutant OsRFPH2-16C188A E3 ligase did not exhibit any activity. We constructed OsRFPH2-16-overexpressing (OX-2 and OX-4) and CRISPR/Cas9-mediated OsRFPH2-16-knockout (KO-4 and KO-16) plants and evaluated their salt responses. Under salt stress, OsRFPH2-16-knockout plants exhibited improved salt tolerance, characterized by low Na+ accumulation, high non-antioxidant content, and dynamic changes in the expression levels of Na+ transporter genes, compared with wild-type and OsRFPH2-16-overexpression plants. The aquaporin OsPIP1;1, an interacting partner, was identified using yeast two-hybridization, bimolecular fluorescence complementation, and pull-down assays. Degradation of OsPIP1;1 by the E3 ligase OsRFPH2-16 via the 26S proteasome system was confirmed through an in vitro degradation assay with the inhibitor MG132. These findings support that the E3 ligase functions as a negative regulator, leading to reduced Na+ accumulation in salt stress responses.

Oryza↗
PubMed1976

Role of deoxyribonucleic acid ligase in a doxyribonucleic acid membrane fraction extracted from pneumococci.

Deoxyribonucleic acid (DNA) ligase has been detected in a DNA membrane fraction extracted from Pneumococcus. The specific activity of the enzyme in this fraction is 10-fold greater than in the remaining cell extract. It remains firmly bound (with other enzymes) to the complex after a purification procedure in which a considerable percentage of the macromolecules are dissociated. The ligase acts in two ways in the DNA membrane fraction in vitro. One, it catalyzes the linkage of small-molecular-weight pieces of newly synthesized DNA into heavier-molecular-weight DNA strands as shown by others (M Gellert, 1976; R. Okazaki, A. Sugino, S. Hirose, T. Okazaki, Y. Imae, R. Kainuma-Kuroda, T. Ogawa, M. Arisawa, and Y. Kurosowa, 1973; B. Olivera and I. Lehman, 14; and A. Sugino, S. Hirose, and R. Okazaki, 1972) and, two, it protects DNA from degradation by deoxyribonucleases. This latter effect is due to a competition between the ability of the nucleases to degrade DNA and the ability of DNA ligase to seal the nicks produced by these degradative enzymes. The ligase acts cooperatively with other enzymes in the DNA membrane fraction to synthesize DNA.

Adenosine Triphosphate↗
PubMed1975

A deoxyribonucleic acid ligase from nuclei of rat liver. Purification and properties.

A DNA ligase has been extensively purified from nuclei of rat livers. The ligase seals single strand nicks in DNA with any of the four usual bases on either the 3 or 5 sides. It requires ATP and a divalent cation (Mg-2plus or Mn-2plus) for activity. At low Mg-2plus concentrations the activity is greatly stimulated by a variety of monovalent cations. Relatively small excesses of either monovalent or divalent cation above the amounts which give maximal activity lead to inhibition of activity. Poly(G) and poly(I) inhibit ligase activity; several other polyribonucleotides are not inhibitory. Low concentrations of inorganic pyrophosphate are inhibitory. The molecular weight of the ligase is estimated from gel filtration to be about 10 times 10-4.

Adenosine Triphosphate↗
PubMed1978

Joining of 3'-modified oligonucleotides by T4 RNA ligase. Synthesis of a heptadecanucleotide corresponding to the bases 61--77 from Escherichia coli tRNAfMet.

Chemically synthesized fragments corresponding to the 3' end of tRNAfMet from Escherichia coli were joined by T4-induced RNA ligase to yield a heptadecanucleotide (bases 61--77). The 3' terminus of C-C-A was modified by introduction of the ethoxymethylidene group to prevent intra- and intermolecular self-joining reactions at the 3' end. The terminal trimer was phosphorylated using polynucleotide kinase and joined to C-A-A with RNA ligase. The hexamer [C-A-A-C-C-A(ethoxymethylidene)] corresponding to bases 72--77 was obtained in a yield of 60%. An undecanucleotide (bases 61--71) which had been synthesized in a yield of 34% by similar enzymatic joining of U-C-C-G-G to pC-C-C-C-C-G was allowed to react with the 5'-phosphorylated hexamer (bases 72--77) using an excess of RNA ligase to yield the heptadecanucleotide U-C-C-G-G-C-C-C-C-C-G-C-A-A-C-C-A (bases 61--77). The product was identified by homochromatography and nearest neighbor analysis.

Base Sequence↗
PubMed1977

Dinucleoside pyrophosphate are substrates for T4-induced RNA ligase.

RNA ligase isolated from bacteriophage T4-infected Escherichia coli will utilize a number of different compounds with the general structure Ado-5'PP-X as substrates in an ATP-independent reaction. The P-X portions of these molecules are transferred to the 3'-hydroxyl of an oligoribonucleotide to form a phosphodiester bond, and the Ado-5'P (AMP) portion is released. AMP, CMP, GMP, UMP, dTMP, NMN, alphaNMN, reduced NMN, FMN, Rib-5P, phosphopantetheine, and cyanoethylphosphate all have been added to [Cyd-3H](Ap)3C from their corresponding AMP adducts. Contrary to the relative lack of specificity of RNA ligase for the P-X -group added, the failure of NADP+, deamino-NAD+, epsilonNCD+, epsilon NAD and CoA to react indicates that the enzyme shows a high degree of selectivity for the AMP portion of the substrate. The diversity of chemical groups that can be efficiently added suggests that this reaction of RNA ligase will prove useful for the modification of the 3' ends of RNA molecules.

Coliphages↗