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PubMed2000

Structural and mechanistic conservation in DNA ligases.

DNA ligases are enzymes required for the repair, replication and recombination of DNA. DNA ligases catalyse the formation of phosphodiester bonds at single-strand breaks in double-stranded DNA. Despite their occurrence in all organisms, DNA ligases show a wide diversity of amino acid sequences, molecular sizes and properties. The enzymes fall into two groups based on their cofactor specificity, those requiring NAD(+) for activity and those requiring ATP. The eukaryotic, viral and archael bacteria encoded enzymes all require ATP. NAD(+)-requiring DNA ligases have only been found in prokaryotic organisms. Recently, the crystal structures of a number of DNA ligases have been reported. It is the purpose of this review to summarise the current knowledge of the structure and catalytic mechanism of DNA ligases.

Adenosine Triphosphate↗
PubMed2005

A colorimetric method for point mutation detection using high-fidelity DNA ligase.

The present study reported proof-of-principle for a genotyping assay approach that can detect single nucleotide polymorphisms (SNPs) through the gold nanoparticle assembly and the ligase reaction. By incorporating the high-fidelity DNA ligase (Tth DNA ligase) into the allele-specific ligation-based gold nanoparticle assembly, this assay provided a convenient yet powerful colorimetric detection that enabled a straightforward single-base discrimination without the need of precise temperature control. Additionally, the ligase reaction can be performed at a relatively high temperature, which offers the benefit for mitigating the non-specific assembly of gold nanoparticles induced by interfering DNA strands. The assay could be implemented via three steps: a hybridization reaction that allowed two gold nanoparticle-tagged probes to hybrid with the target DNA strand, a ligase reaction that generates the ligation between perfectly matched probes while no ligation occurred between mismatched ones and a thermal treatment at a relatively high temperature that discriminate the ligation of probes. When the reaction mixture was heated to denature the formed duplex, the purple color of the perfect-match solution would not revert to red, while the mismatch gave a red color as the assembled gold nanoparticles disparted. The present approach has been demonstrated with the identification of a single-base mutation in codon 12 of a K-ras oncogene that is of significant value for colorectal cancers diagnosis, and the wild-type and mutant type were successfully scored. To our knowledge, this was the first report concerning SNP detection based on the ligase reaction and the gold nanoparticle assembly. Owing to its ease of operation and high specificity, it was expected that the proposed procedure might hold great promise in practical clinical diagnosis of gene-mutant diseases.

Cell Line↗
PubMed2005

Template-independent ligation of single-stranded DNA by T4 DNA ligase.

T4 DNA ligase is one of the workhorses of molecular biology and used in various biotechnological applications. Here we report that this ligase, unlike Escherichia coli DNA ligase, Taq DNA ligase and Ampligase, is able to join the ends of single-stranded DNA in the absence of any duplex DNA structure at the ligation site. Such nontemplated ligation of DNA oligomers catalyzed by T4 DNA ligase occurs with a very low yield, as assessed by quantitative competitive PCR, between 10(-6) and 10(-4) at oligonucleotide concentrations in the range 0.1-10 nm, and thus is insignificant in many molecular biological applications of T4 DNA ligase. However, this side reaction may be of paramount importance for diagnostic detection methods that rely on template-dependent or target-dependent DNA probe ligation in combination with amplification techniques, such as PCR or rolling-circle amplification, because it can lead to nonspecific background signals or false positives. Comparison of ligation yields obtained with substrates differing in their strandedness at the terminal segments involved in ligation shows that an acceptor duplex DNA segment bearing a 3'-hydroxy end, but lacking a 5'-phosphate end, is sufficient to play a role as a cofactor in blunt-end ligation.

DNA 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↗
PubMed1994

4-Hydroxybenzoate-coenzyme A ligase from Rhodopseudomonas palustris: purification, gene sequence, and role in anaerobic degradation.

Anaerobic metabolism of most aromatic acids is initiated by coenzyme A thioester formation. Rhodopseudomonas palustris grows well under anaerobic, phototrophic conditions with many aromatic acids, including benzoate and 4-hydroxybenzoate, as a carbon source. A coenzyme A ligase that reacts with 4-hydroxybenzoate was purified from 4-hydroxybenzoate-grown cells of R. palustris. This enzyme required MgATP, reduced coenzyme A, and 4-hydroxybenzoate, benzoate, or cyclohex-1,4-dienecarboxylate for optimal activity but also used phosphopantetheine, cyclohex-2,5-dienecarboxylate, and 4-fluorobenzoate at lower rates. The 4-hydroxybenzoate-coenzyme A ligase differed in molecular characteristics from a previously described benzoate-coenzyme A ligase from R. palustris, and the two ligases did not cross-react immunologically. The gene encoding the 4-hydroxybenzoate enzyme was cloned and sequenced. The deduced gene product showed about 20% amino acid identity with bacterial coenzyme A ligases involved in aerobic degradation of aromatic acids. An R. palustris mutant carrying a disrupted 4-hydroxybenzoate-coenzyme A ligase gene was unable to grow with 4-hydroxybenzoate under anaerobic conditions, indicating that the enzyme is essential for anaerobic degradation of this compound.

Anaerobiosis↗
PubMed2006

Adenovirus ubiquitin-protein ligase stimulates viral late mRNA nuclear export.

Theadenovirus type 5 (Ad5) E1B-55K and E4orf6 proteins are required together to stimulate viral late nuclear mRNA export to the cytoplasm and to restrict host cell nuclear mRNA export during the late phase of infection. Previous studies have shown that these two viral proteins interact with the cellular proteins elongins B and C, cullin 5, RBX1, and additional cellular proteins to form an E3 ubiquitin-protein ligase that polyubiquitinates p53 and probably one or more subunits of the MRE11-RAD50-NBS1 (MRN) complex, directing their proteasomal degradation. The MRN complex is required for cellular DNA double-strand break repair and induction of the DNA damage response by adenovirus infection. To determine if the ability of E1B-55K and E4orf6 to stimulate viral late mRNA nuclear export requires the ubiquitin-protein ligase activity of this viral ubiquitin-protein ligase complex, we designed and expressed a dominant-negative mutant form of cullin 5 in HeLa cells before infection with wild-type Ad5 or the E1B-55K null mutant dl1520. The dominant-negative cullin 5 protein stabilized p53 and the MRN complex, indicating that it inhibited the viral ubiquitin-protein ligase but had no effect on viral early mRNA synthesis, early protein synthesis, or viral DNA replication. However, expression of the dominant-negative cullin 5 protein caused a decrease in viral late protein synthesis and viral nuclear mRNA export similar to the phenotype produced by mutations in E1B-55K. We conclude that the stimulation of adenovirus late mRNA nuclear export by E1B-55K and E4orf6 results from the ubiquitin-protein ligase activity of the adenovirus ubiquitin-protein ligase complex.

Active Transport, Cell Nucleus↗
PubMed1995

An etoposide-induced block in vaccinia virus telomere resolution is dependent on the virus-encoded DNA ligase.

Etoposide, an inhibitor of the breakage-reunion reaction associated with cellular type II DNA topoisomerases, was shown to inhibit plaque formation of vaccinia virus. This drug had a major effect on the segregation of newly replicated DNA concatemers. Gene expression and the initiation and elongation phases of viral DNA replication were essentially unaffected. Pulsed-field gel electrophoresis of viral DNA replicated in the presence of etoposide revealed two major classes of DNA: the mature monomeric linear genome and DNA that failed to enter the gel (the relative proportions depending on the concentrations of drug). Restriction enzyme analysis showed a severe defect in telomere resolution. In addition, slowly migrating restriction fragments were suggestive of a general recombination defect. We have isolated several etoposide-resistant mutants and used marker rescue and DNA sequencing to localize the resistance-causing mutation to the amino terminus of the viral DNA ligase gene. Inactivation of the DNA ligase also resulted in an etoposide-resistant phenotype, but to a lesser extent. The telomere resolution and segregation defects were corrected both in the drug-resistant mutants and in the DNA ligase knockout mutants. Reinsertion of wild-type or mutant DNA ligase in the viral thymidine kinase locus confirmed the role of the viral DNA ligase in conferring sensitivity not only to etoposide but also to another topoisomerase II inhibitor, 4'-(9-acridinylamino) methanesulphon-m-anisidide (mAMSA). The data suggest that the nonessential DNA ligase is involved in telomere resolution, possibly as part of a general recombinase.

Amino Acid Sequence↗
PubMed2001

The two RNA ligases of the Trypanosoma brucei RNA editing complex: cloning the essential band IV gene and identifying the band V gene.

Kinetoplastid RNA editing is a posttranscriptional insertion and deletion of U residues in mitochondrial transcripts that involves RNA ligase. A complex of seven different polypeptides purified from Trypanosoma brucei mitochondria that catalyzes accurate RNA editing contains RNA ligases of approximately 57 kDa (band IV) and approximately 50 kDa (band V). From a partial amino acid sequence, cDNA and genomic clones of band IV were isolated, making it the first cloned component of the minimal RNA editing complex. It is indeed an RNA ligase, for when expressed in Escherichia coli, the protein autoadenylylates and catalyzes RNA joining. Overexpression studies revealed that T. brucei can regulate of total band IV protein at the level of translation or protein stability, even upon massively increased mRNA levels. The protein's mitochondrial targeting was confirmed by its location, size when expressed in T. brucei and E. coli, and N-terminal sequence. Importantly, genetic knockout studies demonstrated that the gene for band IV is essential in procyclic trypanosomes. The band IV and band V RNA ligases of the RNA editing complex therefore serve different functions. We also identified the gene for band V RNA ligase, a protein much more homologous to band IV than to other known ligases.

Alternative Splicing↗
PubMed2006

Biochemical characterisation of LigN, an NAD+-dependent DNA ligase from the halophilic euryarchaeon Haloferax volcanii that displays maximal in vitro activity at high salt concentrations.

BACKGROUND: DNA ligases are required for DNA strand joining in all forms of cellular life. NAD+-dependent DNA ligases are found primarily in eubacteria but also in some eukaryotic viruses, bacteriophage and archaea. Among the archaeal NAD+-dependent DNA ligases is the LigN enzyme of the halophilic euryarchaeon Haloferax volcanii, the gene for which was apparently acquired by Hfx. volcanii through lateral gene transfer (LGT) from a halophilic eubacterium. Genetic studies show that the LGT-acquired LigN enzyme shares an essential function with the native Hfx. volcanii ATP-dependent DNA ligase protein LigA. RESULTS: To characterise the enzymatic properties of the LigN protein, wild-type and three mutant forms of the LigN protein were separately expressed in recombinant form in E.coli and purified to apparent homogeneity by immobilised metal ion affinity chromatography (IMAC). Non-isotopic DNA ligase activity assays using lambda DNA restriction fragments with 12 bp cos cohesive ends were used to show that LigN activity was dependent on addition of divalent cations and salt. No activity was detected in the absence of KCl, whereas maximum activity could be detected at 3.2 M KCl, close to the intracellular KCl concentration of Hfx. volcanii cells. CONCLUSION: LigN is unique amongst characterised DNA ligase enzymes in displaying maximal DNA strand joining activity at high (> 3 M) salt levels. As such the LigN enzyme has potential both as a novel tool for biotechnology and as a model enzyme for studying the adaptation of proteins to high intracellular salt levels.

DNA Ligases↗
PubMed1991

Deletion analysis of a multifunctional yeast tRNA ligase polypeptide. Identification of essential and dispensable functional domains.

Splicing of tRNA precursors in extracts of Saccharomyces cerevisiae requires the action of two enzymes: a site specific endonuclease and a tRNA ligase. The tRNA ligase contains three distinct enzymatic activities: a polynucleotide kinase, a cyclic phosphodiesterase, and an RNA ligase. The polypeptide also has a high affinity pre-tRNA binding site based on its ability to form stable complexes with pre-tRNA substrates. To investigate the organization of functional enzymatic and binding elements within the polypeptide a series of defined tRNA ligase gene deletions were constructed and corresponding proteins were expressed in Escherichia coli as fusions with bacterial dihydrofolate reductase (DHFR). The DHFR/ligase derivative proteins were then efficiently purified by affinity chromatography. The complete ligase fusion protein retained enzymatic and binding activities which were unaffected by the presence of the DHFR segment. Examination of tRNA ligase deletion derivatives revealed that the amino-terminal region was required for adenylylation, while the carboxyl-terminal region was sufficient for cyclic phosphodiesterase activity. Deletions within the central region affected kinase activity. Pre-tRNA binding activity was not strictly correlated with a distinct enzymatic domain. A DHFR/ligase-derived protein lacking kinase activity efficiently joined tRNA halves. We postulate that this variant utilizes a novel RNA ligation mechanism.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1982

Enzymic synthesis of lignin precursors. Purification and properties of 4-coumarate:CoA ligase from cambial sap of spruce (Picea abies L.).

4-Coumarate:CoA ligase was purified from cambial sap of spruce (Picea abies). A 1627-fold purification of the enzyme with a yield of 37% was achieved by a six-step procedure including dye-ligand chromatography. Isozymes of the ligase were not detected. The enzyme has an Mr of about 63 000 and is a single polypeptide chain. Ferulic, 4-coumaric and caffeic acids are efficient substrates for the ligase. In contrast to some ligases from angiosperms, the ligase from spruce (gymnosperm) does not activate sinapic acid. The substrate specificity of the ligase is consistent with the lignin composition of spruce.

Coenzyme A Ligases↗
PubMed1993

Novel activity of a yeast ligase deletion polypeptide. Evidence for GTP-dependent tRNA splicing.

Yeast tRNA ligase possesses multiple activities which are required for the joining of tRNA halves during the tRNA splicing process: cyclic phosphodiesterase, kinase, adenylylate synthetase, and ligase. A deletion polypeptide of a dihydrofolate reductase-ligase fusion protein, designated DAC, was previously shown to join tRNA halves although ATP-dependent kinase activity was not measurable in the assay used. We describe here a characterization of the mechanism of joining used by DAC and the structure of the tRNA product. DAC produces a joined tRNA and a splice junction with a structure identical to that produced by DAKC, the full-length dihydrofolate reductase-ligase fusion. Furthermore, DAC can use GTP as the sole cofactor in the joining reaction, in contrast to DAKC, which can only complete splicing in the presence of ATP. Both enzymes exhibit GTP-dependent kinase activity at 100-fold greater efficiency than with ATP. These results suggest that a potential function for the center domain of tRNA ligase (missing in DAC) is to provide structural integrity and aid in substrate interactions and specificity. They also support the hypothesis that ligase may prefer to use two different cofactors during tRNA splicing.

Base Sequence↗
PubMed2003

Sepsis upregulates the gene expression of multiple ubiquitin ligases in skeletal muscle.

Muscle wasting during sepsis reflects increased expression and activity of the ubiquitin-proteasome proteolytic pathway and is at least in part mediated by glucocorticoids. The ubiquitination of proteins destined to be degraded by the proteasome is regulated by multiple enzymes, including ubiquitin ligases. We tested the hypothesis that sepsis upregulates the gene expression of the newly described ubiquitin ligases, MuRF1 and atrogin-1/MAFbx. Sepsis was induced in rats by cecal ligation and puncture. Control rats were sham-operated. In some experiments, rats were treated with the glucocorticoid receptor antagonist RU 38486 before induction of sepsis. At various time points after induction of sepsis, mRNA levels for MuRF1 and atrogin-1/MAFbx were determined in extensor digitorum longus muscles by real-time PCR. Sepsis resulted in a 10-16-fold increase in gene expression of the ubiquitin ligases studied here. These changes were much greater than those observed previously for another ubiquitin ligase, E3alpha, in muscle during sepsis. Treatment of rats with RU 38486 prevented the sepsis-induced increase in mRNA levels for MuRF1 and atrogin-1/MAFbx, suggesting that glucocorticoids participate in the upregulation of these genes in muscle during sepsis. The present results lend further support to the concept that the ubiquitin-proteasome pathway plays an important role in sepsis-induced muscle proteolysis and suggest that multiple ubiquitin ligases may participate in the development of muscle wasting during sepsis.

Animals↗
PubMed2001

The infected cell protein 0 of herpes simplex virus 1 dynamically interacts with proteasomes, binds and activates the cdc34 E2 ubiquitin-conjugating enzyme, and possesses in vitro E3 ubiquitin ligase activity.

The infected cell protein 0 (ICP0) of herpes simplex virus 1, a promiscuous transactivator shown to enhance the expression of genes introduced into cells by infection or transfection, interacts with numerous cellular proteins and has been linked to the disruption of ND10 and degradation of several proteins. ICP0 contains a RING finger domain characteristic of a class of E3 ubiquitin ligases. We report that: (i) in infected cells, ICP0 interacts dynamically with proteasomes and is bound to proteasomes in the presence of the proteasome inhibitor MG132. Also in infected cells, cdc34, a polyubiquitinated E2 ubiquitin-conjugating enzyme, exhibits increased ICP0-dependent dynamic interaction with proteasomes. (ii) In an in vitro substrate-independent ubiquitination system, the RING finger domain encoded by exon 2 of ICP0 binds cdc34, whereas the carboxyl-terminal domain of ICP0 functions as an E3 ligase independent of the RING finger domain. The results indicate that ICP0 can act as a unimolecular E3 ubiquitin ligase and that it promotes ubiquitin-protein ligation and binds the E2 cdc34. It differs from other unimolecular E3 ligases in that the domain containing the RING finger binds E2, whereas the ligase activity maps to a different domain of the protein. The results also suggest that ICP0 shuttles between nucleus and cytoplasm as a function of its dynamic interactions with proteasomes.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2003

TIP120A associates with cullins and modulates ubiquitin ligase activity.

The cullin-containing ubiquitin-protein isopeptide ligases (E3s) play an important role in regulating the abundance of key proteins involved in cellular processes such as cell cycle and cytokine signaling. They have multisubunit modular structures in which substrate recognition and the catalysis of ubiquitination are carried out by distinct polypeptides. In a search for proteins involved in regulation of cullin-containing E3 ubiquitin ligases we immunopurified CUL4B-containing complex from HeLa cells and identified TIP120A as an associated protein by mass spectrometry. Immunoprecipitation of cullins revealed that all cullins tested specifically interacted with TIP120A. Reciprocal immunoaffinity purification of TIP120A confirmed the stable interaction of TIP120A with cullin family proteins. TIP120A formed a complex with CUL1 and Rbx1, but interfered with the binding of Skp1 and F-box proteins to CUL1. TIP120A greatly reduced the ubiquitination of phosphorylated IkappaBalpha by SCF(beta-TrCP) ubiquitin ligase. These results suggest that TIP120A functions as a negative regulator of SCF E3 ubiquitin ligases and may modulate other cullin ligases in a similar fashion.

Amino Acid Sequence↗
PubMed1987

Influence of polyethylene glycol on the ligation reaction with calf thymus DNA ligases I and II.

High concentrations of the nonspecific macromolecule polyethylene glycol 6000 (PEG 6000) enabled DNA ligases I and II from calf thymus to catalyze intermolecular blunt-end ligation of duplex DNA. Intermolecular cohesive-end ligation with these enzymes was markedly stimulated in the presence of 10-16% (w/v) PEG 6000. The effect of PEG 6000 (4-16%) on the sealing of single-stranded breaks in duplex DNA with DNA ligases I and II was not appreciably stimulatory but rather inhibitory. PEG 6000 (15%) enhanced more twofold the rate of DNA ligase II-AMP complex formation, but moderately suppressed the rate of formation of DNA ligase 1-AMP complex. Polyamines and KCl inhibited blunt-end and cohesive-end ligations with DNA ligases I and II in the presence of PEG 6000.

Animals↗
PubMed1995

2-Amino-3-ketobutyrate-CoA ligase from beef liver mitochondria: an NMR spectroscopic study of low-barrier hydrogen bonds of a pyridoxal 5'-phosphate-dependent enzyme.

A study of protons associated with low-barrier hydrogen bonds in 2-amino-3-ketobutyrate-CoA ligase (AKB-ligase, EC 2.3.1.29) by NMR is reported. Three resonances are observed in the range of delta H = 15-20 ppm when the NMR spectrum of AKB-ligase is recorded at 600 MHz. These low-barrier hydrogen bonds are associated respectively with a side chain proton, the PLP pyridinium ring nitrogen proton, and the PLP Schiff base proton at the active site of the ligase. The pyridinium proton has been assigned a chemical shift of 19.10 ppm and the Schiff base proton 14.90 ppm. The third low-barrier hydrogen bond associated proton resonating at 16.20 ppm is assigned to a proton of a side chain group. All three resonances disappear when pyridoxal phosphate is removed from the ligase. Consistent with NOE coupling, the side chain group proton should be close to the proton of the Schiff base nitrogen of the pyridoxal 5'-phosphate. The effects of temperature, pH, substrate, and NOE on the three resonances are also studied, in order to assign the protons. The three low-barrier hydrogen bonds described in this report may serve to anchor the cofactor in the active site of 2-amino-3-ketobutyrate-CoA ligase.

Acetyltransferases↗
PubMed1996

Inhibition of human DNA ligase I activity by zinc and cadmium and the fidelity of ligation.

Heavy metals, including zinc (Zn) and cadmium (Cd), are potentially important genotoxic agents in our environment. Here we report that human DNA ligase I, the major form of the enzyme in replicative cells, is a target for Zn and Cd ions. ZnCl2 at 0.8 mM caused complete inhibition of DNA ligase I activity, whereas only 0.04 mM CdCl2 was required to achieve a similar effect. Both metals affected all three steps of the reaction, namely, the formation of ligase-AMP intermediate, the transfer of the AMP to DNA and the ligation reaction that succeeds the formation of the AMP-DNA complex. Unlike F-ara-ATP and the natural protein inhibitor of DNA ligase-I, these metals may affect different domains of the enzyme. Moreover, these metal ions did not increase the rate of misligation of F-ara-A-modified DNA or mismatched DNA substrates, but considerable misligation was observed for the T:C mispairing. These data support the notion of high fidelity of the human DNA ligases and that the major action of these metal ions on the enzyme is their inhibitory function.

Adenosine Monophosphate↗