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PubMed1994

Aberrant DNA repair and DNA replication due to an inherited enzymatic defect in human DNA ligase I.

Two missense mutations in different alleles of the DNA ligase I gene have been described in a patient (46BR) with immunodeficiencies and cellular hypersensitivity to DNA-damaging agents. One of the mutant alleles produces an inactive protein, while the other encodes an enzyme with some residual activity. A subline of identical phenotype that is homozygous (or hemizygous) for the mutant allele encoding this partially active enzyme has facilitated characterization of the enzymatic defect in 46BR. This subline retains only 3 to 5% of normal DNA ligase I activity. The intermediates in the ligation reaction, DNA ligase I-AMP and nicked DNA-AMP, accumulate in vitro and in vivo. The defect of the 46BR enzyme lies primarily in conversion of nicked DNA-AMP into the final ligated DNA product. Assays of DNA repair in 46BR cell extracts and of DNA replication in permeabilized cells have clarified functional roles of DNA ligase I. The initial rate of ligation of Okazaki fragments during DNA replication is apparently normal in 46BR cells, but 25 to 30% of the fragments remain in low-molecular-weight form for prolonged times. DNA base excision repair by 46BR cell extracts shows a delay in ligation and an anomalously long repair patch size that is reduced upon addition of purified normal DNA ligase I.

Adenosine Monophosphate↗
PubMed2002

Distinct functions of two RNA ligases in active Trypanosoma brucei RNA editing complexes.

Trypanosome RNA editing is a unique U insertion and U deletion process that involves cycles of pre-mRNA cleavage, terminal U addition or U removal, and religation. This editing can occur at massive levels and is directed by base pairing of trans-acting guide RNAs. Both U insertion and U deletion cycles are catalyzed by a single protein complex that contains only seven major proteins, band I through band VII. However, little is known about their catalytic functions, except that band IV and band V are RNA ligases and genetic analysis indicates that the former is important in U deletion. Here we establish biochemical approaches to distinguish the individual roles of these ligases, based on their distinctive ATP and pyrophosphate utilization. These in vitro analyses revealed that both ligases serve in RNA editing. Band V is the RNA editing ligase that functions very selectively to seal in U insertion (IREL), while band IV is the RNA editing ligase needed to seal in U deletion (DREL). In combination with our earlier findings about the cleavage and the U-addition/U-removal steps of U deletion and U insertion, these results show that all three steps of these editing pathways exhibit major differences and suggest that the editing complex could have physically separate regions for U deletion and U insertion.

Adenosine Triphosphate↗
PubMed1993

The ubiquitin-ligase system in Trypanosoma brucei brucei.

The question of whether the African trypanosome Trypanosoma brucei brucei utilizes a ubiquitin-ligase system to conjugate the 8,500-dalton protein ubiquitin to other proteins has not been investigated. Using 125I-labeled ubiquitin and gel electrophoresis (sodium dodecyl sulfate and acetic acid, urea, Triton X-100), we looked for the incorporation of label into proteins larger than ubiquitin to determine ubiquitin-ligase system activity in cytosolic and nuclear lysates of long slender, intermediate, and short stumpy bloodstream-form trypanosomes. We present data suggesting that there is cytosolic activity of a ubiquitin-ligase system in all three bloodstream forms of T. brucei brucei. There are indications that the three bloodstream forms of T. brucei brucei differ in their cytosolic ubiquitin-ligase system activity. Our assay showed no activity of this system in the nucleus of T. brucei brucei. Further studies on the ubiquitin-ligase system in T. brucei brucei may define differences between the three bloodstream forms, the parasite, and its host, leading to development of novel chemotherapeutic strategies.

Analysis of Variance↗
PubMed2002

Replication failure, genome instability, and increased cancer susceptibility in mice with a point mutation in the DNA ligase I gene.

DNA ligase I has a key role in DNA replication in the joining together of short replication intermediates. We used gene targeting to introduce a point mutation into the mouse DNA ligase I gene that was present in a human cancer patient with immunodeficiency and a cellular accumulation of DNA replication intermediates. Mutant mice grew more slowly and showed hematopoietic defects at critical stages at which the demands for DNA replication were highest. In the spleen and thymus of mutant mice, the accumulation of a sub-G1, but nonapoptotic, population was observed that we believe may represent cells with single-strand DNA breaks. In mutant bone marrow, occasional DNA replication failure was observed. The level of genome instability was significantly elevated in the spleens of DNA ligase I mutant mice and, because we have found no evidence for any DNA repair defect associated with DNA ligase I deficiency, we believe that this may result directly from the accumulation of replication intermediates. Mutant mice showed an increased incidence of spontaneous cancers with a diverse range of epithelial tumors, particularly cutaneous adnexal tumors that are rare in mice. The origin of the tumors from generalized genome instability, rather than the inactivation of one key control gene, should make DNA ligase I mutant mice a useful model to investigate the relationship between genome instability and cancer in humans.

Animals↗
PubMed1988

Association of a possible DNA ligase deficiency with T-cell acute leukemia.

DNA ligase activities were determined in blood samples of 431 different cases of lymphoblastic and nonlymphoblastic leukemia. Less activity was observed in samples from lymphoblastic patients. RNA translation together with ligase immunoprecipitation experiments show that in T-cell acute lymphoblastic leukemic cells, no ligase is translated. This deficiency is correlated with the presence of more breaks in the DNA from these kinds of leukemia which results in altered DNA. This DNA can be ligated by the addition of exogenous ligase. This is the first demonstration of a ligase deficiency in leukemic human cells. These results are discussed in terms of chromosome abnormalities and rearrangements of genes coding for enzymes involved in DNA replication and repair.

Acute Disease↗
PubMed1989

dATP-mediated inhibition of DNA ligase by 2'-deoxycoformycin in T and B cell leukemia.

2'-Deoxycoformycin (dCF), a potent adenosine deaminase inhibitor, has been reported to display greater toxicity for T than for B lymphoblasts. Since this compound can block DNA replication and since this effect is mediated by the intracellular ATP/dATP balance, its possible effect on DNA ligase was investigated. dCF at relatively low concentrations (1 microM), in association with dATP (100 microM), is a strong inhibitor of DNA ligase in T blasts, whereas it has no significant effect in B blasts at this concentration. The AMP-ligase complex is the target of the observed inhibition because the combined presence of the inhibitor and dATP results in a more stable dAMP-ligase complex. Because of this observation and of the greater adenosine deaminase activity observed in T cells, the dATP mediated dCF inhibition of ligase might be the crucial replication target of T cell toxicity. These observations are discussed in terms of T immunodeficiencies including Graft Versus Host Disease and related syndromes.

Adenosine Triphosphate↗
PubMed1987

Differentiation of thymocytes during human ontogeny: stage-specific DNA ligase in relation to terminal deoxynucleotidyl transferase, cell size and surface antigen.

The activities of two forms (7.5 and 5.5 S) of DNA ligase and of terminal deoxynucleotidyl transferase (TdT) have been studied in human thymocytes at different ages from 20 weeks pre-natal to 37 years after birth. Thymocytes have been selected on the basis of relative size and antigenicity (CD3, OKT3 immunofluorescence) with the cell sorter. For DNA ligases, three kinds of cells can be distinguished: (i) large antigenically negative cells of 20-week fetus, expressing only the 7.5 S enzyme; (ii) large antigenically positive cells without ligase activity; (iii) smaller antigenically positive cells, expressing only the 5.5 S enzyme. This last form of enzyme is found after birth. With respect to TdT expressed in OKT3- 5 micron cells and to OKT3+ thymocytes, it is observed that 5.5 S DNA ligase is found in a thymocyte population distinct from cells expressing TdT. Therefore, these results allow us to consider the 5.5 S DNA ligase activity as an additional functional marker for thymocyte maturation in humans.

Adolescent↗
PubMed1984

Studies on RNA ligase activity in the brain and liver cells of mouse.

RNA ligase in eukaryotic mammalian cells was studied by using mouse brain and liver cell extracts as enzyme sources and Oligo A as substrates. RNA ligase activity was determined by measuring the formation of alkaline phosphate-resistant product from 5'-32P-terminated Oligoribonucleotides. Under appropriate conditions, the activity of this enzyme in brain and liver cells may vary between 16-49 mU/ml. The joining way between donor and acceptor is 5'-P----3'-OH. Further studies were carried out by using synthetic UpCpU and 32pNp as substrates and crude enzyme preparations from extracts of cell nuclei of brain and liver as enzyme sources. RNA ligase activity was examined by homochromatography and autoradiography. A clear joining product was demonstrated and then isolated from the reaction mixture by DEAE-Sephadex A25 column chromatography. The eluted fractions were identified by DEAE-cellulose thin layer chromatography. The joining product was hydrolyzed either with KOH or with alkaline phosphatase, the autoradiographic spot of the product disappeared. In this case the joining way between donor and acceptor is 3'-P----5'-OH instead of 5'P----3'-OH. All this indicated that in extracts of mouse brain and liver cells most probably exists some other kind of RNA ligase, which differs from the T4 RNA ligase in the joining way.

Adenosine Triphosphate↗
PubMed1982

Eukaryotic DNA ligase. Purification and properties of the enzyme from bovine thymus, and immunochemical studies of the enzyme from animal tissues.

DNA ligase has been purified to near-homogeneity from the extract of bovine thymus with a yield of 5%. The purified enzyme catalyzed the joining of single-stranded breaks in duplex DNA at a rate of 33 nmol of phosphodiester bonds/min/mg of protein. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis and Ouchterlony double diffusion analysis. The enzyme is composed of a single polypeptide with a molecular weight of about 130,000. The enzyme has a Stokes radius of 52 A, a sedimentation coefficient of about 5 S, and a frictional ratio of 1.6. Apparent Km values for ATP and Mg2+ are 2 microM and 0.9 mM, respectively. Antibody against bovine thymus DNA ligase was prepared by injecting a rabbit with the purified enzyme. Immunochemical titrations revealed that the increased activity of DNA ligase observed after partial hepatectomy of rat and 16-fold higher activity level of mouse Ehrlich tumor cells compared with the host liver are due to a change in the enzyme quantity but not to a change in the catalytic efficiency of the enzyme molecule. Wide variations in the level of DNA ligase activity in extracts from various tissues of rat and mouse were accompanied by proportionate changes in the quantity of immunochemically reactive protein. The antibody inhibited DNA ligase activity from bovine tissues with 20-fold higher efficiency, compared with the enzyme from the rodent tissues. The enzyme activity from chick embryo was unaffected by the antibody.

Animals↗
PubMed1996

Interaction of salicylate and ibuprofen with the carboxylic acid: CoA ligases from bovine liver mitochondria.

Neither salicylate nor ibuprofen was a substrate or inhibitor of the long-chain fatty acid:CoA ligase. In contrast, all three xenobiotic-metabolizing medium-chain fatty acid:CoA ligases (XL-I, XL-II, and XL-III) had activity toward salicylate. The K(m) value for salicylate was similar for all three forms (2 to 3 microM), but XL-II and XL-III had higher activity at Vmax. For ibuprofen, only XL-III catalyzed its activation, and it had a K(m) for ibuprofen of 36 microM. Studies of salicylate inhibition of XL-I, XL-II, and XL-III revealed that it inhibited the benzoate activity of all three forms with K1 values of ca. 2 microM, which is in agreement with the K(m) values obtained with salicylate as substrate. Kinetic analysis revealed that salicylate conjugation by all three forms is characterized by substrate inhibition when salicylate exceeds ca. 20 microM. Substrate inhibition was more extensive with XL-I and XL-III. Previous work on the ligases employed assay concentrations of salicylate in the range of 0.1 to 1.0 mM, which are clearly inhibitory, particularly toward XL-I and XL-III. Thus, activity was not properly measured in previous studies, which accounts for the fact that salicylate conjugation was only found with one form, which is most likely XL-II since it has the highest Vmax activity and shows the least amount of substrate inhibition. Studies with ibuprofen indicated that it inhibited XL-I, XL-II, and XL-III, with KI values being in the range of 75-125 microM. The short-chain ligase was inhibited by both salicylate and ibuprofen with KI values of 93 and 84 microM, respectively. It was concluded that pharmacological doses of salicylate, but not ibuprofen, will affect the metabolism of medium-chain fatty acids and carboxylic acid xenobiotics and that the previously described mitochondrial ibuprofen:CoA ligase activity is attributable to XL-III.

Animals↗
PubMed2002

Rejoining of DNA strand breaks by T4 DNA ligase in mammalian cells.

We have tested the ability of T4 DNA ligase to rejoin radiation-induced DNA strand breaks in living hamster cells (CHO-K1, EM9, xrs-5). T4 DNA ligase was introduced into cells by electroporation prior to x-irradiation. Single- and double-strand breaks were measured by the alkaline comet assay technique, and double-strand breaks (DSBs) were evaluated by the pulsed-field gel electrophoresis method. In the comet assay, the three cell lines showed reduced tail moments following pretreatment with T4 DNA ligase, both directly after irradiation and after repair incubation for 4 h. Similarly, the results obtained from pulsed-field gel electrophoresis showed reduced DSB frequencies after pretreatment with T4 DNA ligase. We conclude that exogeneous T4 ligase contributes to rejoining of radiation-induced strand breaks.

Animals↗
PubMed1991

Vaccinia virus DNA ligase is nonessential for virus replication: recovery of plasmids from virus-infected cells.

The essentiality of the vaccinia virus DNA ligase gene, SalF 15R, for virus growth was tested by insertional mutagenesis. A plasmid containing E. coli gpt inserted within a large deletion in the DNA ligase gene was transfected into vaccinia virus-infected cells and recombinant viruses selected by three cycles of plaque purification in the presence of mycophenolic acid (MPA). Surprisingly, in some isolates, which replicated in a manner indistinguishable from wild type (WT) virus, the WT gene was replaced by the gpt allele, demonstrating that the DNA ligase gene is nonessential for growth in cultured cells. In other isolates the entire plasmid was integrated into the virus genome by a single crossover event and a functional copy of the DNA ligase was retained. Southern blot analyses of the latter, drug-resistant viruses indicated extra DNA fragments, of sizes inconsistent with predicted viral structures, which represent the plasmid products of homologous recombination. Hirt extracts from cells infected with such multiply plaque purified virus isolates yielded plasmids that produced ampicillin-resistant colonies after transformation of E. coli. These plasmids were of two structures, representing either the original plasmid used for transfection, or a plasmid containing the WT ligase gene rescued by recombination with the virus genome. Similarly, insertional mutagenesis of the vaccinia virus thymidine kinase (TK) gene with gpt yielded plasmids containing mutant or wild type TK alleles when recombinant viruses were selected in MPA. Such plasmids were not isolated when TK minus viruses were selected in 5-bromodeoxyuridine (BUdR).

Animals↗
PubMed1991

Cloning, overexpression and nucleotide sequence of a thermostable DNA ligase-encoding gene.

Thermostable DNA ligase has been harnessed for the detection of single-base genetic diseases using the ligase chain reaction [Barany, Proc. Natl. Acad. Sci. USA 88 (1991) 189-193]. The Thermus thermophilus (Tth) DNA ligase-encoding gene (ligT) was cloned in Escherichia coli by genetic complementation of a ligts 7 defect in an E. coli host. Nucleotide sequence analysis of the gene revealed a single chain of 676 amino acid residues with 47% identity to the E. coli ligase. Under phoA promoter control, Tth ligase was overproduced to greater than 10% of E. coli cellular proteins. Adenylated and deadenylated forms of the purified enzyme were distinguished by apparent molecular weights of 81 kDa and 78 kDa, respectively, after separation via sodium dodecyl sulfate-polyacrylamide-gel electrophoresis.

Amino Acid Sequence↗
PubMed2006

h-Goliath, paralog of GRAIL, is a new E3 ligase protein, expressed in human leukocytes.

In Drosophila, the RING finger protein d-Goliath was originally identified as a transcription factor involved in the embryo mesoderm formation [Bouchard, M.L., Cote, S., 1993. The Drosophila melanogaster developmental gene g1 encodes a variant zinc-finger-motif protein. Gene 125, 205-209]. In mouse, the m-Goliath mRNA level was shown to be increased in growth factor withdrawal-induced apoptosis of myeloid cells [Baker, S.J., Reddy, E.P., 2000. Cloning of murine G1RP, a novel gene related to Drosophila melanogaster g1. Gene 248, 33-40]. Due to its putative function of transcription factor in apoptosis, we cloned the human cDNA for h-Goliath and characterized the expression of the protein in blood and bone marrow cells. The human protein of 419 aa (44 kDa) contains a protease-associated domain, a transmembrane domain and a RING-H2 motif. This structure classifies h-Goliath as a new member of a human family of ubiquitin ligases with GRAIL (gene related to anergy in lymphocytes) as founder. This E3 ligase controls the development of T cell clonal anergy by ubiquitination [Anandasabapathy, N., Ford, G.S., Bloom, D., Holness, C., Paragas, V., Seroogy, C., Skrenta, H., Hollenhorst, M., Fathman, C.G., Soares, L., 2003. GRAIL: an E3 ubiquitin ligase that inhibits cytokine gene transcription is expressed in anergic CD4+ T cells. Immunity 18, 535-547]. In vitro ubiquitination studies support the E3 ubiquitin ligase activity of h-Goliath. In human, the protein is expressed under 3 isoforms, a major one at 28 kDa and two others at 46 and 55 kDa. These proteins come from a common precursor (44 kDa) as we observed using in vitro transcription-translation. Using immunohistochemistry on blood or bone marrow smears, of healthy or leukemia samples, we found that the protein expression was restricted to the cytoplasm of progenitors and fully differentiated leukocyte populations. We did not observe any modification of h-Goliath expression or localization in leukemia. In these cells, this new E3 ubiquitin ligase protein does not seem associated with a differentiation state of the cell or with apoptosis.

Amino Acid Motifs↗
PubMed2004

High resolution crystal structure of a key editosome enzyme from Trypanosoma brucei: RNA editing ligase 1.

Trypanosomatids are causative agents of several devastating tropical diseases such as African sleeping sickness, Chagas' disease and leishmaniasis. There are no effective vaccines available to date for treatment of these protozoan diseases, while current drugs have limited efficacy, significant toxicity and suffer from increasing resistance. Trypanosomatids have several remarkable and unique metabolic and structural features that are of great interest for developing new anti-protozoan therapeutics. One such feature is "RNA editing", an essential process in these pathogenic protozoa. Transcripts for key trypanosomatid mitochondrial proteins undergo extensive post-transcriptional RNA editing by specifically inserting or deleting uridylates from pre-mature mRNA in order to create mature mRNAs that encode functional proteins. The RNA editing process is carried out in a approximately 1.6 MDa multi-protein complex, the editosome. In Trypanosoma brucei, one of the editosome's core enzymes, the RNA editing ligase 1 (TbREL1), has been shown to be essential for survival of both insect and bloodstream forms of the parasite. We report here the crystal structure of the catalytic domain of TbREL1 at 1.2 A resolution, in complex with ATP and magnesium. The magnesium ion interacts with the beta and gamma-phosphate groups and is almost perfectly octahedrally coordinated by six phosphate and water oxygen atoms. ATP makes extensive direct and indirect interactions with the ligase via essentially all its atoms while extending its base into a deep pocket. In addition, the ATP makes numerous interactions with residues that are conserved in the editing ligases only. Further away from the active site, TbREL1 contains a unique loop containing several hydrophobic residues that are highly conserved among trypanosomatid RNA editing ligases which may play a role in protein-protein interactions in the editosome. The distinct characteristics of the adenine-binding pocket, and the absence of any close homolog in the human genome, bode well for the design of selective inhibitors that will block the essential RNA ligase function in a number of major protozoan pathogens.

Adenosine Triphosphate↗
PubMed2004

Adenylation-dependent conformation and unfolding pathways of the NAD+-dependent DNA ligase from the thermophile Thermus scotoductus.

In the last few years, an increased attention has been focused on NAD(+)-dependent DNA ligases. This is mostly due to their potential use as antibiotic targets, because effective inhibition of these essential enzymes would result in the death of the bacterium. However, development of an efficient drug requires that the conformational modifications involved in the catalysis of NAD(+)-dependent DNA ligases are understood. From this perspective, we have investigated the conformational changes occurring in the thermophilic Thermus scotoductus NAD(+)-DNA ligase upon adenylation, as well as the effect of cofactor binding on protein resistance to thermal and chemical (guanidine hydrochloride) denaturation. Our results indicate that cofactor binding induces conformational rearrangement within the active site and promotes a compaction of the enzyme. These data support an induced "open-closure" process upon adenylation, leading to the formation of the catalytically active enzyme that is able to bind DNA. These conformational changes are likely to be associated with the protein function, preventing the formation of nonproductive complexes between deadenylated ligases and DNA. In addition, enzyme adenylation significantly increases resistance of the protein to thermal denaturation and GdmCl-induced unfolding, establishing a thermodynamic link between ligand binding and increased conformational stability. Finally, chemical unfolding of deadenylated and adenylated enzyme is accompanied by accumulation of at least two equilibrium intermediates, the molten globule and premolten globule states. Maximal populations of these intermediates are shifted toward higher GdmCl concentrations in the case of the adenylated ligase. These data provide further insights into the properties of partially folded intermediates.

Adenine↗
PubMed2002

Making AppDNA using T4 DNA ligase.

5('),5(')-Adenylyl pyrophosphoryl DNA (AppDNA) contains a high-energy pyrophosphate linkage and can be exploited as an activated DNA substrate to derive new DNA enzymes for carrying out various DNA modification reactions. For this reason, enzymatic synthesis of AppDNA is highly desirable. AppDNA is a known intermediate in DNA ligase mediated DNA ligation reactions, but rarely accumulates under normal reaction conditions. Here we report that T4 DNA ligase can quantitatively convert 5(')-phosphoryl DNA donor into AppDNA in the absence of acceptor DNA but in the presence of a template DNA that contains at least one unpaired nucleotide opposite the 5(')-phosphoryl DNA donor site. This adenylylation behavior of T4 DNA ligase is not observed with Thermus aquaticus (Taq) and Escherichia coli DNA ligases. We further found that a donor-template duplex of 11-bp in length is required by T4 DNA ligase for the formation of AppDNA.

Adenosine Monophosphate↗
PubMed2005

Bi-substrate kinetic analysis of an E3-ligase-dependent ubiquitylation reaction.

Little is known about the kinetic mechanism of E3 ubiquitin ligases. This work describes basic methodology to investigate the kinetic mechanism of E3 ubiquitin ligases. The method used steady state, bi-substrate kinetic analysis of an E3 ligase-catalyzed monoubiquitylation reaction using ubiquitin-conjugated E2 (E2ub) and a mutant IkappaBalpha as substrates to evaluate whether the E3-catalyzed ubiquitin transfer from E2ub to protein substrate was sequential, meaning both substrates bound before products leaving, or ping pong, meaning that ubiquitin-conjugated E2 would bind, transfer ubiquitin to the E3, and debind before binding of protein substrate. The method requires the E3 reaction to be rate limiting and at steady state. This was accomplished through optimization of the conditions to ensure that the E3-dependent transfer of ubiquitin from E2ub to substrate was rate limiting. We observed a sequential bi-substrate E3-dependent ubiquitylation reaction on using E2UBCH7 and IkappaBalphaSS32/36EE (IkappaBalphaee as substrates and a partially purified Jurkat cell lysate as a source for the E3 ligase activity). The sequential bi-substrate kinetic mechanism is consistent with the formation of a ternary complex among E2UBCH7, IkappaBalphaSS32/36EE, and E3 before the transfer of ubiquitin from E2UBCH7 to IkappaBalphaSS32/36EE. The described method should be of use to characterize the kinetic mechanism of other E3 ligase-catalyzed ubiquitylation reactions.

Kinetics↗