Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “LIGASES”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 649 records · Page 36Linked to original sources
PubMed1992

Purification and characterization of benzoyl-CoA ligase from a syntrophic, benzoate-degrading, anaerobic mixed culture.

The benzoyl-CoA ligase from an anaerobic syntrophic culture was purified to homogeneity. It had a molecular mass of around 420 kDa and consisted of seven or eight subunits of 58 kDa. The temperature optimum was 37-40 degrees C, the optimum pH around 8.0 and optimal activity required 50-100 mM TRIS-HCl buffer, pH 8.0 and 3-7 mM MgCl2; MgCl2 in excess of 10 mM was inhibitory. The activation energy for benzoate was 11.3 kcal/mol. Although growth occurred only with benzoate as a carbon source, the benzoyl-coenzyme A (CoA) ligase formed benzoyl-CoA esters with benzoate, 2-, 3- and 4-fluorobenzoate, picolinate, nicotinate and isonicotinate. Acetate was activated to acetyl-CoA by an acetyl-CoA synthetase. The Km values for benzoate, 2-, 3- and 4-fluorobenzoate were 0.04, 0.28, 1.48 and 0.32 mM, the Vmax values 1.05, 1.0, 0.7 and 0.98 units (U)/mg, respectively. For reduced CoA (CoA-SH) a Km of 0.07 mM and a Vmax of 1.05 U/mg and for ATP a Km of 0.16 mM and a Vmax of 1.08 U/mg was determined. Benzoate activation was inhibited by more than 6 mM ATP, presumably by pyrophosphate generation from ATP. The inhibition constant (Ki) for pyrophosphate was 5.7 mM. No homology of the N-terminal amino acid sequence with that of a 2-aminobenzoyl-CoA ligase of a denitrifying Pseudomonas sp. was found.

Acetate-CoA Ligase↗
PubMed1986

Induction of yeast DNA ligase genes in exponential and stationary phase cultures in response to DNA damaging agents.

UV-irradiation of stationary phase cells of Saccharomyces cerevisiae and Schizosaccharomyces pombe leads to a 9-fold and 90-fold increase in transcript levels from the respective DNA ligase genes CDC9 and CDC17, whereas exponential cells show only 3-fold and 2-fold increases. Induction of CDC9 after MMS treatment and gamma-irradiation was also observed by using a CDC9-lacZ translational fusion and assaying for beta-galactosidase. Surprisingly, irradiation of S. cerevisiae induces only a 50% increase in DNA ligase itself, probably reflecting the extremely high in vivo stability of the enzyme. The UV-induction of ligase may be part of a "fail-safe" mechanism which, together with the enzyme stability, ensures adequate supplies of this essential enzyme.

DNA Damage↗
PubMed1985

An improved assay for DNA ligase reveals temperature-sensitive activity in cdc9 mutants of Saccharomyces cerevisiae.

A sensitive and quantitative assay for DNA ligase has been developed which is suitable for the analysis of crude cell extracts of yeast. The assay is sufficiently sensitive to detect the low levels of DNA ligase activity remaining in cdc9 mutants of Saccharomyces cerevisiae. Indeed, we have been able to show that this residual activity is temperature-sensitive, thus establishing finally that CDC9 is the structural gene for DNA ligase.

Base Sequence↗
PubMed1992

Benzamide potentiation of the cytotoxicity of bifunctional galactitol [correction of galacticol] in resistant P388 leukemia correlates with inhibition of DNA ligase II.

Benzamide (BA) enhances the cytotoxicity of 1,2:5,6-dianhydrogalactitol (DAG) in resistant P388 leukemia cell lines but not in the sensitive parent line. To examine the reason for this difference in response, we carried out an alkaline elution assay using proteinase K to study DNA interstrand cross-linking. At early time points, equal concentrations of DAG produced the same level of interstrand cross-linking (ICL) in the resistant and sensitive P388 leukemic cells, although marked differences were observed in their cytotoxicity toward the two cell lines. In the sensitive cells, neither the amount of DNA cross-linking nor the cytotoxicity changed during the observation period (38 h) in either the presence or the absence of BA. In contrast, the elution rate of the DNA of DAG-treated resistant cells increased with time and had reached the control levels by 38 h. However, when these cells were postincubated with BA for 38 h, the elution rate of DNA was much faster than that observed for the untreated resistant cells, indicating an accumulation of DNA single-strand breaks (SSB). The SSB accumulation caused by BA was associated with an inhibition of the activity of ligase II enzyme, which was stimulated when resistant cells were treated with DAG alone. The potentiating effect of BA on the resistant cells can thus be related to the inhibiting action of BA on the DNA-rejoining enzyme, ligase II. The lack of sensitization by BA of the DAG-treated parent cell line may be attributable to the absence of DNA-SSB formation, which is necessary for ligase II activation through the stimulation of poly(ADP-ribose) synthesis.

Animals↗
PubMed1985

Synthesis and purification of oligoribonucleotides using T4 RNA ligase and reverse-phase chromatography.

T4 RNA ligase has been used to construct a series of defined oligoribonucleotides. Hexamer or pentamer blocks were synthesized first by multiple additions of mononucleotide diphosphates to trimers with T4 RNA ligase and removal of the terminal phosphate with alkaline phosphatase; inhibitors of the ligase were removed by passing the sample over a 1-ml reverse-phase octadecasilyl column. The two nucleotide blocks were then ligated to give undecamers. Yields for the individual ligations ranged from 85 to 100% for acceptors lacking uridines and at least 70% for those containing uridines. The overall yield of the undecamer relative to the starting trimers was about 10%. Each round of ligation averaged about 8 h; the time required to synthesize each undecamer was 1 to 2 weeks. Optimization of the steps to achieve this is described in detail.

Autoradiography↗
PubMed1985

Substrate specificity of fatty-acyl-CoA ligase in liver microsomes.

The substrate specificity of fatty-acyl-CoA ligase in liver microsomes has been studied in a system in which fatty acids are present initially as complexes with unilamellar vesicles of phosphatidylcholine. The latter were prepared by cosonication of phospholipids and different fatty acids. As compared with previous studies of the enzyme the activity of acyl-CoA ligase is several-fold higher for assays carried out with fatty acid substrates added as components of a bilayer. This was true for all fatty acids studied. Also as compared with data reported previously in the literature there was a systematic relationship between the structure of fatty acids, activity at Vmax for synthesis of acyl-CoA and avidity of binding to the ligase. Activity at Vmax was greatest for lauric acid and decreased with increasing chain length. The apparent avidity of enzyme for fatty acids was greatest for octanoic acid and decreased as chain length increased.

Adenosine Triphosphate↗
PubMed1981

Joining of yeast alanine transfer ribonucleic acid half molecules to form a whole molecule by T4 RNA ligase.

Covalent joining of the two half molecules of tRNAAla by T4 RNA ligase to form a reconstituted whole molecule was investigated. The two half molecules consisting, respectively, of residues 1-35 and 36-75 were prepared by partial degradation of tRNAAla with RNAase T1. The 5'-half molecule was treated with alkaline phosphatase to remove the 3'-terminal phosphate group, and the 5'-OH group of the 3'-half molecule was phosphorylated with [gamma-32P]ATP by polynucleotide kinase. The two terminal nucletides to be joined were identified as Guo and Cyd. Prior to the covalent joining reaction, the two modified half molecules in an equimolar mixture were annealed, and the rejoined half molecules, separated by gel electrophoresis, served as the substrate for T4 RNA ligase. Optimum conditions for this ligation, such as RNA ligase concentration, pH, Mg2+ concentration, reaction temperature and time of reaction, were investigated. Under the optimum conditions a yield of about 70% joining of the reconstituted whole molecule was obtained as shown by gel electrophoresis, resistance to hydrolysis by alkaline phosphatase, nearest neighbour analysis and alanine acdeptor activity.

Alanine↗
PubMed1983

DNA ligase activity in crude extracts of fibroblasts and lymphocytes.

DNA ligase activity was determined in crude cell extracts using a new assay which measures the retention of double stranded circular phage lambda DNA on nitrocellulose filters, and allows accurate determinations of the enzyme activity with cell concentration corresponding to 0.1 microgram of proteins. Using this assay, we show that the DNA ligase activity varies greatly among mammalian cell lines. The higher activity is found in actively growing fibroblasts where it is stimulated by dimethyl sulfate pretreatment of the cells, whereas the low activity measured in resting lymphocytes is not modified by dimethyl sulfate. The DNA ligase activity correlates with the cells sensitivity towards ionizing radiations.

Animals↗
PubMed1983

Spermidine and spermine stimulate the activity of T4-DNA ligase.

When the ability of T4-DNA ligase from E. coli NM 989 to form higher molecular weight polymers from linearized plasmid pJDB 207 was followed, it was observed that physiological concentrations (0.5 to 1.0 mM) of spermidine and spermine greatly stimulated the formation of these polymers. The effect had a strict specificity since 1,3-diaminopropane, putrescine (1,4-diaminobutane) and N1-acetylspermidine neither stimulated nor inhibited this activity of DNA ligase. The structural analogues of spermidine, methyl bis(guanylhydrazone) and 1,1'-[(methylethanediylidene)dinitrilo]bis(3-aminoguanidine) totally abolished the stimulatory effect of spermidine on T4-DNA ligase without affecting the enzyme's basal activity.

DNA Ligases↗
PubMed1990

Identification of DNA ligase I related polypeptides in three different human cells.

Partial purification of the DNA ligase from three human tissues (liver, thymus and lymphoblasts) revealed that each cell type contains several different polypeptides bearing a DNA ligase I activity. Their apparent molecular weights estimated after SDS PAGE, 130 kDa, 100 kDa and 80 kDa, are in agreement with previous reports. These polypeptides are related by proteolysis to a single higher molecular weight protein of 200 kDa which does not show DNA ligase activity but that could be a preprotein.

Adenosine Monophosphate↗
PubMed1985

Molecular duality of DNA ligase in axolotl corresponds to distinctive transcriptional information.

Based upon the use of specific antibodies and sucrose gradient sedimentation analysis, the present work describes the use of the post-transcriptional equipment of the urodele egg to compare the information contained in two RNA samples extracted from respectively liver and activated axolotl eggs. It is shown that besides the normal DNA ligase activity present in the host Pleurodeles eggs, RNA can translate for the specific carried information revealing a difference between the two samples. Moreover, unlike in nuclear transplantation, the homologous DNA ligases are not mutually exclusive. These observations give a new convincing support of the genetic basis of the molecular duality of DNA ligases.

Ambystoma↗
PubMed1983

RNA ligase in bacteria: formation of a 2',5' linkage by an E. coli extract.

Ligase activity was detected in extracts of Escherichia coli, Clostridium tartarivorum, Rhodospirillum salexigens, Chromatium gracile, and Chlorobium limicola. Ligase was measured by joining of tRNA halves produced from yeast IVS-containing tRNA precursors by a yeast endonuclease. The structure of tRNATyr halves joined by an E. coli extract was examined. The ligated junction is resistant to nuclease P1 and RNAase T2 but sensitive to venom phosphodiesterase and alkaline hydrolysis, consistent with a 2',5' linkage. The nuclease-resistant junction dinucleotide comigrates with authentic (2',5') APA marker in thin-layer chromatography. The phosphate in the newly formed phosphodiester bond is derived from the pre-tRNA substrate. The widespread existence of a bacterial ligase raises the possibility of a novel class of RNA processing reactions.

Escherichia coli↗
PubMed1992

Evaluation of the ligase chain reaction (LCR) for the detection of point mutations.

The ligase chain reaction (LCR) was evaluated as an amplification method for an in vivo mutation assay. Specifically, the ligase was tested for its ability to selectively amplify a DNA sequence mutated at a single base, in the presence of an excess of wild-type DNA. As a model template a 370-bp DNA fragment of the mouse Ha-ras protooncogene containing an A to T mutation at the second position of codon 61 was used. With the commercially available ligase Ampligase (Epicenter), 250 molecules of mutant fragments could be detected by an enzyme-linked immunoassay with digoxigenin marker (giving a theoretical detection limit of 1 target gene per 10(4) copies of genome). In the analysis of mixtures with corresponding wild-type DNA fragments, a 1:1 mixture resulted in a clearly stronger signal than control samples lacking wild-type and mutant DNA. However, the signal obtained from a 100-fold dilution of the mutant DNA with wild-type DNA could not be distinguished from the background noise. In this particular form, LCR lacks sufficient selectivity to be applied to an in vivo situation, where the ratio of mutant to wild-type DNA sequences might be expected to lie around 1:10(6).

Animals↗
PubMed1983

Kinetic studies on the reaction catalyzed by DNA ligase from calf thymus.

Kinetic analysis of the reaction catalyzed by calf thymus DNA ligase (EC 6.5.1.1) has been carried out using [5'-32P]nicked DNA as substrate. The results of initial velocity and product inhibition studies indicate that the ligase reaction is likely to proceed through the 'uni-uni uni-bi ping-pong' mechanism. The order of substrate addition and product release is as follows: ATP, PPi, nicked DNA, sealed DNA and 5'-AMP. The true Km values for ATP and for nicked DNA (5'-phosphoryl ends) were 2 microM and 0.11 microM, respectively. The turnover number was estimated to be 7 sealing events per min. dATP was an inhibitor competitive with ATP (Ki = 25 microM). The addition of 0.5 mM spermine or 5 mM spermidine resulted in an increase in the apparent Km for nicked DNA as well as in the apparent V, whereas 0.1 M KCl increased only the apparent Km for nicked DNA. Neither polyamine nor KCl affected the apparent Km for ATP. The ligase reaction was not significantly affected by aphidicolin and various phosphate compounds tested.

Adenine Nucleotides↗
PubMed1987

Normal DNA ligase activity in a gamma-ray-sensitive Chinese hamster mutant.

A Chinese hamster cell mutant (XR-1) was previously described that is extremely deficient in the repair of double-strand DNA breaks produced by gamma-irradiation during the sensitive G1--early-S period and somewhat deficient in repair of gamma-ray-induced single-strand DNA breaks. To determine whether a deficiency in DNA ligase activity might underlie the biochemical defect, protein extracts from mutant and parental cells were examined for their ability to ligate single- and double-strand breaks in DNA. The kinetics of ligation of single 5'-phosphate-3'-hydroxyl breaks in double-stranded DNA were the same in protein extracts from both cells. After separation of protein extracts by gel-filtration chromatography, the percentage of activity in the large and small molecular forms of DNA ligase was also similar in the two cells. Finally, protein extracts prepared from exponentially growing or G1-synchronized mutant and parental cells were equal in their ability to ligate blunt-end DNA substrates. These data suggest that a deficiency in DNA ligase is not the cause of the repair defect in the XR-1 mutant cell.

Animals↗
PubMed1989

Specificity of the nick-closing activity of bacteriophage T4 DNA ligase.

Bacteriophage T4 DNA ligase effectively joins two adjacent, short synthetic oligodeoxyribonucleotides (oligos), as guided by complementary oligo, plasmid and genomic DNA templates. When a single bp mismatch exists at either side of the ligation junction, the efficiency of the enzyme to ligate the two oligos decreases. Mismatch ligation is approximately five-fold greater if the mismatch occurs at the 3' side rather than at the 5' side of the junction. During mismatch ligation the 5' adenylate of the 3' oligo accumulates in the reaction. The level of the adenylate formation correlates closely with the level of the mismatch ligation. Both mismatch ligation and adenylate formation are suppressed at elevated temperatures and in the presence of 200 mM NaCl or 2-5 mM spermidine. The apparent Km for the oligo template in the absence of salt is 0.05 microM, whereas the Km increases to 0.2 microM in the presence of 200 mM of NaCl. In this report, we demonstrate these properties of T4 DNA ligase for oligo pairs complementary to the beta-globin gene at the sequence surrounding the single bp mutation responsible for sickle-cell anemia. Because of the highly specific nature of the nick-closing reaction, ligation of short oligos with DNA ligase can be used to distinguish two DNA templates differing by a single nucleotide.

Adenosine Monophosphate↗
PubMed2006

Distinct ubiquitin-ligase complexes define convergent pathways for the degradation of ER proteins.

Many misfolded endoplasmic reticulum (ER) proteins are eliminated by ERAD, a process in which substrates are polyubiquitylated and moved into the cytosol for proteasomal degradation. We have identified in S. cerevisiae distinct ubiquitin-ligase complexes that define different ERAD pathways. Proteins with misfolded ER-luminal domains use the ERAD-L pathway, in which the Hrd1p/Hrd3p ligase forms a near stoichiometric membrane core complex by binding to Der1p via the linker protein Usa1p. This core complex associates through Hrd3p with Yos9p, a substrate recognition protein in the ER lumen. Substrates with misfolded intramembrane domains define a pathway (ERAD-M) that differs from ERAD-L by being independent of Usa1p and Der1p. Membrane proteins with misfolded cytosolic domains use the ERAD-C pathway and are directly targeted to the Doa10p ubiquitin ligase. All three pathways converge at the Cdc48p ATPase complex. These results lead to a unifying concept for ERAD that may also apply to mammalian cells.

Adenosine Triphosphatases↗
PubMed2004

Solution structure and DNA binding of the zinc-finger domain from DNA ligase IIIalpha.

DNA ligase IIIalpha carries out the final ligation step in the base excision repair (BER) and single strand break repair (SSBR) mechanisms of DNA repair. The enzyme recognises single-strand nicks and other damage features in double-stranded DNA, both through the catalytic domain and an N-terminal domain containing a single zinc finger. The latter is homologous to other zinc fingers that recognise damaged DNA, two in the N terminus of poly(adenosine-ribose)polymerase and three in the N terminus of the Arabidopsis thaliana nick-sensing DNA 3'-phosphoesterase. Here, we present the solution structure of the zinc-finger domain of human DNA ligase IIIalpha, the first structure of a finger from this group. It is related to that of the erythroid transcription factor GATA-1, but has an additional N-terminal beta-strand and C-terminal alpha-helix. Chemical shift mapping using a DNA ligand containing a single-stranded break showed that the DNA-binding surface of the DNA-ligase IIIalpha zinc finger is substantially different from that of GATA-1, consistent with the fact that the two proteins recognise very different features in the DNA. Likely implications for DNA binding are discussed.

Amino Acid Sequence↗