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At least 451 records · Page 25Linked to original sources
PubMed1997

Characterization of an ATP-dependent DNA ligase encoded by Chlorella virus PBCV-1.

We report that Chlorella virus PBCV-1 encodes a 298-amino-acid ATP-dependent DNA ligase. The PBCV-1 enzyme is the smallest member of the covalent nucleotidyl transferase superfamily, which includes the ATP-dependent polynucleotide ligases and the GTP-dependent RNA capping enzymes. The specificity of PBCV-1 DNA ligase was investigated by using purified recombinant protein. The enzyme catalyzed efficient strand joining on a singly nicked DNA in the presence of magnesium and ATP (Km, 75 microM). Other nucleoside triphosphates or deoxynucleoside triphosphates could not substitute for ATP. PBCV-1 ligase was unable to ligate across a 2-nucleotide gap and ligated poorly across a 1-nucleotide gap. A native gel mobility shift assay showed that PBCV-1 DNA ligase discriminated between nicked and gapped DNAs at the substrate-binding step. These findings underscore the importance of a properly positioned 3' OH acceptor terminus in substrate recognition and reaction chemistry.

Adenosine Monophosphate↗
PubMed1995

Guide RNA-mRNA chimeras, which are potential RNA editing intermediates, are formed by endonuclease and RNA ligase in a trypanosome mitochondrial extract.

RNA editing in kinetoplast mitochondrial transcripts involves the insertion and/or deletion of uridine residues and is directed by guide RNAs (gRNAs). It is thought to occur through a chimeric intermediate in which the 3' oligo(U) tail of the gRNA is covalently joined to the 3' portion of the mRNA at the site being edited. Chimeras have been proposed to be formed by a transesterification reaction but could also be formed by the known mitochondrial site-specific nuclease and RNA ligase. To distinguish between these models, we studied chimera formation in vitro directed by a trypanosome mitochondrial extract. This reaction was found to occur in two steps. First, the mRNA is cleaved in the 3' portion of the editing domain, and then the 3' fragment derived from this cleavage is ligated to the gRNA. The isolated mRNA 3' cleavage product is a more efficient substrate for chimera formation than is the intact mRNA, inconsistent with a transesterification mechanism but supporting a nuclease-ligase mechanism. Also, when normal mRNA cleavage is inhibited by the presence of a phosphorothioate, normal chimera formation no longer occurs. Rather, this phosphorothioate induces both cleavage and chimera formation at a novel site within the editing domain. Finally, levels of chimera-forming activity correlate with levels of mitochondrial RNA ligase activity when reactions are conducted under conditions which inhibit the ligase, including the lack of ATP containing a cleavable alpha-beta bond. These data show that chimera formation in the mitochondrial extract occurs by a nuclease-ligase mechanism rather than by transesterification.

Animals↗
PubMed2003

Trypanosoma brucei RNA editing complex: band II is structurally critical and maintains band V ligase, which is nonessential.

Maturation of Trypanosoma brucei mitochondrial mRNA involves massive posttranscriptional insertion and deletion of uridine residues. This RNA editing utilizes an enzymatic complex with seven major proteins, band I through band VII. We here use RNA interference (RNAi) to examine the band II and band V proteins. Band II is found essential for viability; it is needed to maintain the normal structure of the editing complex and to retain the band V ligase protein. Previously, band III was found essential for certain activities, including maintenance of the editing complex and retention of the band IV ligase protein. Thus, band II and band V form a protein pair with features analogous to the band III/band IV ligase pair. Since band V is specific for U insertion and since band IV is needed for U deletion, their parallel organization suggests that the editing complex has a pseudosymmetry. However, unlike the essential band IV ligase, RNAi to band V has only a morphological but no growth rate effect, suggesting that it is stimulatory but nonessential. Indeed, in vitro analysis of band V RNAi cell extract demonstrates that band IV can seal U insertion when band V is lacking. Thus, band IV ligase is the first activity of the basic editing complex shown able to serve in both forms of editing. Our studies also indicate that the U insertional portion may be less central in the editing complex than the corresponding U deletional portion.

Amino Acid Sequence↗
PubMed2005

A novel RING-type ubiquitin ligase breast cancer-associated gene 2 correlates with outcome in invasive breast cancer.

The RING finger family of proteins possess ubiquitin ligase activity and play pivotal roles in protein degradation and receptor-mediated endocytosis. In this study, we examined whether the breast cancer-associated gene 2 (BCA2), a novel RING domain protein, has E3 ubiquitin ligase activity and investigated its expression status in breast tumors. The full-length BCA2 gene was cloned from the human breast cancer cell line MDA-MB-468. It encodes an open reading frame of 304 amino acids and contains a RING-H2 domain. BCA2 maps to chromosome 1q21.1, a region known to harbor cytogenetic aberrations in breast cancers. We found that the BCA2 protein has an intrinsic autoubiquitination activity, the hallmark of E3 ligases, whereas mutant RING protein is not autoubiquitinated. This indicates that the BCA2 ubiquitin ligase activity is dependent on the RING-H2 domain. Using tissue microarrays and immunohistochemistry, we found strong to intermediate BCA2 staining in 56% of 945 invasive breast cancers cases, which was significantly correlated with positive estrogen receptor status [odds ratio (OR), 1.51; P = 0.004], negative lymph node status (OR, 0.73; P = 0.02), and an increase in disease-free survival for regional recurrence (OR, 0.45; P = 0.03). Overexpression of BCA2 increased proliferation and small interfering RNA inhibited growth of T47D human breast cancer cells and NIH3T3 mouse cells. The autoubiquitination activity of BCA2 indicates that it is a novel RING-type E3 ligase. Its association with clinical measures and its effects on cell growth indicate that BCA2 may be important for the ubiquitin modification of proteins crucial to breast carcinogenesis and growth.

Adult↗
PubMed2006

Differing effects of T4 DNA ligase in the modulation of the damage induced in mammalian cells by either X-rays or restriction endonucleases.

BACKGROUND: Of the different lesions induced by X-rays, DNA double-strand breaks (DSBs) are considered the main cause of chromosomal aberrations and cell death. Restriction endonucleases (REs) induce only DNA DSBs and have frequently been used to mimic the effects of ionizing radiation in the study of DNA damage and repair. METHODS: The present work makes use of clonogenic and cytogenetic assays to study the effect of T4 DNA ligase on modulating the damage induced by either X-rays or an RE (MspI) that produces breaks with cohesive ends. A CHO cell line defective in ligase III activity (EM9) and its corresponding parental line (AA8) were used. RESULTS: Our results show that T4 DNA ligase increased cell survival and decreased chromosomal aberrations in cells treated with MspI, suggesting that most RE-induced DSBs can be repaired by a simple ligation. This enzyme was, however, unable to promote repair of the DNA damage induced by X-rays. Analysis of the ratios of exchange-type aberrations to chromatid break-type aberrations indicated that T4 ligase increased misrejoining of the DNA damage induced by X-rays. The results were similar in EM9 and AA8 cells, although the effect was greater in the cells deficient in DNA strand break rejoining. In addition, depending on whether the end strand break structure is 3'-hydroxyl and 5'-phosphoryl (REs) or more complex (X-rays), T4 DNA ligase could either promote the correct repair or, conversely, increase misrejoining. CONCLUSION: The present results confirm the idea that DNA DSBs induced by cohesive cutting RE are repaired by different mechanisms than those induced by X-rays causing cell lethality.

Animals↗
PubMed2005

RNA-directed construction of structurally complex and active ligase ribozymes through recombination.

RNA-directed recombination can be used to catalyze a disproportionation reaction among small RNA substrates to create new combinations of sequences. But the accommodation of secondary and tertiary structural constraints in the substrates by recombinase ribozymes has not been explored. Here, we show that the Azoarcus group I intron can recombine oligoribonucleotides to construct class I ligase ribozymes, which are catalytically active upon synthesis. The substrate oligonucleotides, ranging in size from 58 to 104 nucleotides (nt), along with the 152-nt ligase ribozymes they reconstitute, can contain significant amounts of secondary structure. However, substrate recognition by the Azoarcus ribozyme depends on the existence of a single accessible CAU triplet for effective recombination. A biphasic temperature reaction profile was designed such that the sequential recombination/ligation events could take place in a thermocycler without human intervention. A temperature-dependent pH shift of the reaction buffer contributes to the success of the net reaction. When the substrate for the ligase ribozyme is introduced into the reaction mixture, as much as 11% can be observed being converted to product by the recombined ligase in the same reaction vessel. Recombination followed by ligation can also occur under isothermal conditions at 37 degrees C. Tertiary structure formation of the ligase upon construction can provide some protection from cleavage by the Azoarcus ribozyme when compared to the constituent substrates. These data suggest that RNA-directed recombination can, in fact, articulate complex ribozymes, and that there are logical rules that can guide the optimal placement of the CAU recognition sequence.

Adenosine Monophosphate↗
PubMed2006

Efficient RNA 5'-adenylation by T4 DNA ligase to facilitate practical applications.

We describe a simple procedure for RNA 5'-adenylation using T4 DNA ligase. The 5'-monophosphorylated terminus of an RNA substrate is annealed to a complementary DNA strand that has a 3'-overhang of 10 nucleotides. Then, T4 DNA ligase and ATP are used to synthesize 5'-adenylated RNA (5'-AppRNA), which should find use in a variety of practical applications. In the absence of an acceptor nucleic acid strand, the two-step T4 DNA ligase mechanism is successfully interrupted after the adenylation step, providing 40%-80% yield of 5'-AppRNA after PAGE purification with few side products (the yield varies with RNA sequence). Optimized reaction conditions are described for 5'-adenylating RNA substrates of essentially any length including long and structured RNAs, without need for sequestration of the RNA 3'-terminus to avoid circularization. The new procedure is applicable on the preparative nanomole scale. This 5'-adenylation strategy using T4 DNA ligase is a substantial improvement over our recently reported adenylation method that uses T4 RNA ligase, which often leads to substantial amounts of side products and requires careful optimization for each RNA substrate. Efficient synthetic access to 5'-adenylated RNA will facilitate a range of applications by providing substrates for in vitro selection; by establishing a new protocol for RNA 5'-capping; and by providing an alternative approach for labeling RNA with (32)P or biophysical probes at the 5'-terminus.

Adenosine Triphosphate↗
PubMed2004

Ubiquitin-protein ligases--novel therapeutic targets?

Intracellular protein degradation is a tightly regulated process that in many cases is controlled by protein ubiquitylation. The ubiquitin pathway is a major route by which cells not only remove normal proteins at the appropriate time but also abnormally folded normal or mutant, cytoplasmic and membrane, proteins. This has led to a major impetus to identify constituents of the pathway. The key components that regulate substrate ubiquitylation are the ubiquitin-protein ligases. Ligases come in many forms, from single proteins to very large multiprotein complexes. Specificity of targeting can be modulated by the requirement for post-translational modifications such as phosphorylation, hydroxylation or oxidation of the substrate and, in some cases, the ligase itself. The requirement for substrate modification prior to ubiquitylation allows the same ligase to target different substrates within the same cell at different times. Abnormal intracellular protein processing is a common feature of many human diseases including neurodegenerative diseases and cancer. It may not represent the causative factor that initiates the disease process but may be a downstream regulator of the toxic effect. These abnormalities often arise from the loss of a key protein-protein interaction. As a consequence, mutated proteins can have very different half-lives from their normal counterparts. This can affect the levels of their activity and/or lead to the formation of protein aggregates (inclusion bodies/aggresomes). In this review, we aim to highlight examples of diseases where abnormal protein ubiquitylation is proposed to be a key regulator of the disease process. The recent success of the proteasome inhibitor Bortezomib (PS-341) for treatment of relapsed, refractory myeloma suggests that the modulation of individual ubiquitin-protein ligase activities with synthetic agents may represent a novel approach that has enormous potential for the treatment of a wide range of diseases.

Cysteine Endopeptidases↗
PubMed1992

Benzoyl-CoA ligase activity in the liver and kidney cortex of weanling guinea pigs treated with various inducers: relationship with hippurate synthesis and carnitine levels.

Recent reports have indicated that guinea pig may be a better model for the study of benzoate conjugation in humans because of the presence of a salicylate-activating ligase. We gave intraperitoneal injections of sodium benzoate, sodium salicylate, sodium methylprednisolone and sodium phenobarbital for 4 consecutive days to groups of weanling guinea pigs before sacrifice. A measurement of benzoyl-CoA ligase activity and in vitro hippurate synthesis was done in liver and kidney cortex homogenates from each treated group at 21 days of age, as compared to an untreated (saline) 21-day control group. The results indicate that benzoyl-CoA ligase in guinea pigs is affected by phenobarbital (liver and kidney; p < 0.05-0.01) and methylprednisolone (liver; p < 0.01). Liver hippurate synthesis was also significantly induced by phenobarbital (p < 0.01). Both these treated groups also had reduced levels of free carnitine in plasma taken from 21-day-old guinea pigs as compared to the untreated control. Sodium benzoate did not have any effect on benzoyl-CoA ligase activity or on in vitro hippurate synthesis. However, sodium salicylate did show some effect on the induction of liver and kidney benzoyl-CoA ligase (not significant) and liver (p < 0.05) and kidney hippurate synthesis. It is important that studies on the guinea pig model be extended for application to children receiving therapy of benzoate and other aromatic acids like p-aminobenzoate or salicylate.

Animals↗
PubMed1992

Yeast tRNA ligase mutants are nonviable and accumulate tRNA splicing intermediates.

We show here that yeast tRNA ligase protein is essential in the cell and participates in joining together tRNA half-molecules resulting from excision of the intron by the splicing endonuclease. A haploid yeast strain carrying a chromosomal deletion of the ligase gene is viable only if ligase protein can be supplied from a plasmid copy of the gene. When synthesis of the plasmid-borne ligase gene is repressed, cells eventually die and accumulate endonuclease cut but unligated half-molecules and intervening sequences. Half-molecules that accumulate appear to be fully end-processed. Two temperature-sensitive ligase mutant strains have been isolated; these strains accumulate a similar set of unligated half-molecules at the nonpermissive temperature.

Chromosome Deletion↗
PubMed1992

Inhibition of T4 DNA ligase activity by (+)-CC-1065: demonstration of the importance of the stiffening and winding effects of (+)-CC-1065 on DNA.

Non-denaturing gel electrophoresis analysis demonstrates that the stiffening and winding effects of (+)-CC-1065 produce unusual proximal and distal inhibition of T4 DNA ligase-catalysed ligation of covalently modified DNA. (+)-CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis. This drug selectively bonds through N3 of adenine in DNA and lies in the minor groove of DNA, reacting in a highly sequence-selective manner. Previous studies (Lee et al., 1991) have shown that (+)-CC-1065 produces bending and winding of DNA. The DNA bending and sequence specificity is mediated by the alkylating 'A' subunit of (+)-CC-1065, while the close van der Waals contacts between the non-alkylating B and C subunits of (+)-CC-1065 and the floor of the minor groove of DNA are responsible for the winding of DNA. Covalent modification of oligomers with (+)-CC-1065 and structurally related drugs leads to preferential inhibition of T4 DNA ligase on the non-covalently modified strand to the 5' side of the covalent adduct site, but enhanced ligation of the covalently modified strand. We speculate that the differential effect on proximal strand ligation is due to a drug-induced winding and helix-stabilizing effect which occurs predominantly to the 5' side of the adduct. In addition to the proximal inhibition of ligation, we also describe a distal inhibition of T4 DNA ligase activity which occurs exclusively with drug-modified oligomers and that, if successful, would result in 180 degrees out-of-phase bent DNA following ligation. In this case, two 25 mers or a 21 plus a 29 mer are inhibited from ligation when modified with (+)-CC-1065. This distal ligation is unique to (+)-CC-1065 and its analogs that cause stiffening of the DNA helix. The (+)-CC-1065-induced stiffening effect was demonstrated using a circularization assay and was found to be associated with the close van der Waals contacts between the inside edge of (+)-CC-1065 and the floor of the minor groove, and also to the benzofuran moiety of (+)-ABC" (Adozelesin), a (+)-CC-1065 analog. We conclude from these studies that the DNA-winding and helix-stabilizing effects of these drug molecules can dramatically affect the efficiency of proximal ligation mediated by T4 DNA ligase, and the unusual helix-stiffening effect of (+)-CC-1065, (+)-AB'C' and (+)-ABC" can stabilize the structure of bent DNA formed by drug modification, which results in distal ligase inhibition.

Antibiotics, Antineoplastic↗
PubMed1990

[The role of hydroxyl groups of tyrosine residues in RNA-ligase].

The stability of the AMP-RNA-ligase complex was studied. The complex was found to be stable for at least two weeks upon storage at 0 degrees. Approximately 50% of the initial radioactivity was retained after storage at 9 degrees for 49 days. The formation of adenosine in the complex decomposition products suggested the possible involvement of the tyrosine hydroxyl group in the formation of AMP-RNA-ligase complexes. In order to study the structure of the enzyme active site, the hydroxyl groups of the RNA-ligase tyrosine residues were modified with N-acetyl-imidazole. It was found that four out of 25 tyrosine residues are exceptionally susceptible to chemical modification with resultant complete suppression of the enzyme ability to form a complex with ATP. The kinetic parameters (Km, V) of native and modified RNA-ligase were determined and compared. A hypothetical scheme of the tyrosine hydroxyl group participation in RNA-ligase-catalyzed reactions is proposed.

Adenosine Monophosphate↗
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↗
PubMed1995

Salvaging recombinants from low-efficiency ligase reactions for more efficient subcloning.

Certain types of ligase reactions can be problematic, such as those involving PCR products, blunt-ends and multiple DNA inserts. A simple PCR-based strategy was developed to overcome cloning difficulties with these inefficient ligase reactions. After an initial ligase reaction, primers complementary to the vector are utilized to amplify the DNA fragment from (the few) successful recombinants in the ligation mixture. This DNA fragment is processed for use in a more conventional and straightforward ligase reaction. We demonstrate the potential of the technique by applying it to a variety of difficult ligase reactions.

Animals↗
PubMed1993

Multiple nucleotide cofactor use by yeast ligase in tRNA splicing. Evidence for independent ATP- and GTP-binding sites.

We have examined multiple cofactor usage by yeast tRNA ligase in splicing in vitro. The ligase mechanism of action requires expenditure of two molar equivalents of nucleotide cofactor per mole of tRNA product. Recent evidence (Westaway, S.K., Belford, H.G., Apostol, B.L., Abelson, J., and Greer, C.L. (1993) J. Biol. Chem. 268, 2435-2443) demonstrated that the ligase-associated kinase activity is more efficient with GTP as cofactor than with ATP. Employing a ligase fusion construct with dihydrofolate reductase (Apostol, B.L., Westaway, S.K., Abelson, J., and Greer, C.L. (1991) J. Biol. Chem. 266, 7445-7455) for purposes of enzyme purification, we performed joining assays demonstrating that ATP and GTP are the most effective combination of cofactors. ATP was essential to the joining reaction, while UTP, CTP, or ATP replaced GTP inefficiently. Specific and functionally independent binding sites were confirmed for ATP and GTP by direct binding measurement. A third site was implicated in UTP- and CTP-ligase interactions. Comparison of binding constants with Kapp values determined for nucleotide-dependent joining suggested both that nucleotide triphosphate binding may be limiting in tRNA joining and that tRNA ligation occurs most efficiently using GTP for the kinase reaction and ATP as the adenylylate synthetase cofactor.

Adenosine Triphosphate↗
PubMed2006

RNA ligase structures reveal the basis for RNA specificity and conformational changes that drive ligation forward.

T4 RNA ligase 2 (Rnl2) and kinetoplastid RNA editing ligases exemplify a family of RNA repair enzymes that seal 3'OH/5'PO(4) nicks in duplex RNAs via ligase adenylylation (step 1), AMP transfer to the nick 5'PO(4) (step 2), and attack by the nick 3'OH on the 5'-adenylylated strand to form a phosphodiester (step 3). Crystal structures are reported for Rnl2 at discrete steps along this pathway: the covalent Rnl2-AMP intermediate; Rnl2 bound to an adenylylated nicked duplex, captured immediately following step 2; and Rnl2 at an adenylylated nick in a state poised for step 3. These structures illuminate the stereochemistry of nucleotidyl transfer and reveal how remodeling of active-site contacts and conformational changes propel the ligation reaction forward. Mutational analysis and comparison of nick-bound structures of Rnl2 and human DNA ligase I highlight common and divergent themes of substrate recognition that can explain their specialization for RNA versus DNA repair.

Adenosine Monophosphate↗
PubMed2003

Parkin is a component of an SCF-like ubiquitin ligase complex and protects postmitotic neurons from kainate excitotoxicity.

Mutations in parkin, which encodes a RING domain protein associated with ubiquitin ligase activity, lead to autosomal recessive Parkinson's disease characterized by midbrain dopamine neuron loss. Here we show that parkin functions in a multiprotein ubiquitin ligase complex that includes the F-box/WD repeat protein hSel-10 and Cullin-1. HSel-10 serves to target the parkin ubiquitin ligase activity to cyclin E, an hSel-10-interacting protein previously implicated in the regulation of neuronal apoptosis. Consistent with the notion that cyclin E is a substrate of the parkin ubiquitin ligase complex, parkin deficiency potentiates the accumulation of cyclin E in cultured postmitotic neurons exposed to the glutamatergic excitotoxin kainate and promotes their apoptosis. Furthermore, parkin overexpression attenuates the accumulation of cyclin E in toxin-treated primary neurons, including midbrain dopamine neurons, and protects them from apoptosis.

Animals↗
PubMed2002

Herpes simplex virus 1-infected cell protein 0 contains two E3 ubiquitin ligase sites specific for different E2 ubiquitin-conjugating enzymes.

Infected cell protein 0 (ICP0) of herpes simplex virus 1, a multifunctional ring finger protein, enhances the expression of genes introduced into cells by infection or transfection, interacts with numerous cellular and viral proteins, and is associated with the degradation of several cellular proteins. Sequences encoded by exon 2 of ICP0 (residues 20-241) bind the UbcH3 (cdc34) ubiquitin-conjugating enzyme, and its carboxy terminus expresses a ubiquitin ligase activity demonstrable by polyubiquitylation of cdc34 in vitro. We report that: (i) The physical interaction of cdc34 and ICP0 leads to its degradation. Thus, substitution of ICP0 aspartate 199 with alanine attenuates the degradation of cdc34 and its binding to the ICP0 ring finger domain. (ii) Substitution of residue 620 reported to abolish the interaction with a ubiquitin-specific protease has no effect on the function of ubiquitin ligase. (iii) ICP0 contains an additional distinct E3 ligase activity specific for the UbcH5a- and UbcH6 E2-conjugating enzymes mapping to the ring finger domain. This is, to our knowledge, the first identification of a viral protein with at least two physically separated E3 ligase activities with different E2 specificities. The results suggest that each activity may target different proteins.

Amino Acid Substitution↗