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At least 469 records · Page 26Linked to original sources
PubMed1987

Structural alterations of DNA ligase I in Bloom syndrome.

Cell lines derived from seven patients with Bloom syndrome all contain a DNA ligase I with unusual properties. Six lines were shown to have a reduced level of this enzyme activity and the residual enzyme was anomalously heat-labile. The seventh line contained a dimeric rather than monomeric form of ligase I. Several cell lines representative of other inherited human syndromes have apparently normal DNA ligases. The data indicate that Bloom syndrome is due to a defect in the structure of DNA ligase I caused by a "leaky" point mutation occurring at one of at least two alternative sites.

Bloom Syndrome↗
PubMed2000

The CUL1 C-terminal sequence and ROC1 are required for efficient nuclear accumulation, NEDD8 modification, and ubiquitin ligase activity of CUL1.

Members of the cullin and RING finger ROC protein families form heterodimeric complexes to constitute a potentially large number of distinct E3 ubiquitin ligases. We report here that the highly conserved C-terminal sequence in CUL1 is dually required, both for nuclear localization and for modification by NEDD8. Disruption of ROC1 binding impaired nuclear accumulation of CUL1 and decreased NEDD8 modification in vivo but had no effect on NEDD8 modification of CUL1 in vitro, suggesting that ROC1 promotes CUL1 nuclear accumulation to facilitate its NEDD8 modification. Disruption of NEDD8 binding had no effect on ROC1 binding, nor did it affect nuclear localization of CUL1, suggesting that nuclear localization and NEDD8 modification of CUL1 are two separable steps, with nuclear import preceding and required for NEDD8 modification. Disrupting NEDD8 modification diminishes the IkappaBalpha ubiquitin ligase activity of CUL1. These results identify a pathway for regulation of CUL1 activity-ROC1 and the CUL1 C-terminal sequence collaboratively mediate nuclear accumulation and NEDD8 modification, facilitating assembly of active CUL1 ubiquitin ligase. This pathway may be commonly utilized for the assembly of other cullin ligases.

Amino Acid Sequence↗
PubMed2000

Opportunistic urine ligase chain reaction screening for sexually transmitted diseases in adolescents seeking care in an urban emergency department.

STUDY OBJECTIVE: Neisseria gonorrhoeae and Chlamydia trachomatis are the most common bacterial sexually transmitted diseases (STDs) in sexually active youth and many infections are asymptomatic or unrecognized. This study used ligase chain reaction assays for determination of prevalence of gonococcal and chlamydial infections in adolescents seeking care at an urban emergency department. METHODS: An unlinked prevalence study was performed with ligase chain reaction tests on voided urine specimens from a convenience sample of adolescents 14 years or older who sought care at the Children's Hospital of Alabama ED. Demographic data and data on care provided in the ED were determined from retrospective chart review of those patients whose urine specimens were tested. RESULTS: Of 282 urine specimens screened, 13.5% (38) yielded positive findings on ligase chain reaction testing for either or both pathogens (20 [7%] positive for N gonorrhoeae, 23 [8%] positive for C trachomatis). In the context of acute care, gonorrhea or chlamydial infection was diagnosed in 5 (1.8%). STD prevalence did not vary significantly by age. Only 39% (15/38) of patients with infections detected by ligase chain reaction testing received potentially effective antibiotics as a result of their urgent care evaluation. CONCLUSION: Many adolescents use the ED for nonurgent care and unsuspected STDs are often missed. Urine ligase chain reaction testing is a sensitive, noninvasive means of detecting STDs by which unsuspected adolescent STD cases can be detected in an ED setting.

Adolescent↗
PubMed2006

The Cullin3 ubiquitin ligase functions as a Nedd8-bound heterodimer.

Cullins are members of a family of scaffold proteins that assemble multisubunit ubiquitin ligase complexes to confer substrate specificity for the ubiquitination pathway. Cullin3 (Cul3) forms a catalytically inactive BTB-Cul3-Rbx1 (BCR) ubiquitin ligase, which becomes functional upon covalent attachment of the ubiquitin homologue neural-precursor-cell-expressed and developmentally down regulated 8 (Nedd8) near the C terminus of Cul3. Current models suggest that Nedd8 activates cullin complexes by providing a recognition site for a ubiquitin-conjugating enzyme. Based on the following evidence, we propose that Nedd8 activates the BCR ubiquitin ligase by mediating the dimerization of Cul3. First, Cul3 is found as a neddylated heterodimer bound to a BTB domain-containing protein in vivo. Second, the formation of a Cul3 heterodimer is mediated by a Nedd8 molecule, which covalently attaches itself to one Cul3 molecule and binds to the winged-helix B domain at the C terminus of the second Cul3 molecule. Third, complementation experiments revealed that coexpression of two distinct nonfunctional Cul3 mutants can rescue the ubiquitin ligase function of the BCR complex. Likewise, a substrate of the BCR complex binds heterodimeric Cul3, suggesting that the Cul3 complex is active as a dimer. These findings not only provide insight into the architecture of the active BCR complex but also suggest assembly as a regulatory mechanism for activation of all cullin-based ubiquitin ligases.

Amino Acid Sequence↗
PubMed1976

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

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

HeLa Cells↗
PubMed1978

Leucine: tRNA Ligase from Cultured Cells of Nicotiana tabacum var. Xanthi: Evidence for de Novo Synthesis and for Loss of Functional Enzyme Molecules.

Leucine:tRNA ligase was assayed in extracts from cultured tobacco (Nicotiana tabacum) XD cells by measuring the initial rate of aminoacylation of transfer RNA with l-[4,5-(3)H]leucine. Transfer RNA was purified from tobacco XD cells after the method of Vanderhoef et al. (Phytochemistry 9: 2291-2304). The buoyant density of leucine:tRNA ligase from cells grown for 100 generations in 2.5 mm [(15)N]nitrate and 30% deuterium oxide was 1.3397. After transfer of cells into light medium (2.5 mm [(14)N]nitrate and 100% H(2)O) the ligase activity increased and the buoyant density decreased with time to 1.3174 at 72 hours after transfer. It was concluded that leucine:tRNA ligase molecules were synthesized de novo from light amino acids during the period of activity increase. The width at half-peak height of the enzyme distribution profiles following isopycnic equilibrium centrifugation in caesium chloride remained constant at all times after transfer into light medium providing evidence for the loss of preexisting functional ligase molecules. It was concluded that during the period of activity increase the cellular level of enzyme activity was determined by a balance between de novo synthesis and the loss of functional enzyme molecules due to either inactivation or degradation.

Journal Article↗
PubMed2005

Characterization of Chlamydia MurC-Ddl, a fusion protein exhibiting D-alanyl-D-alanine ligase activity involved in peptidoglycan synthesis and D-cycloserine sensitivity.

Recent characterization of chlamydial genes encoding functional peptidoglycan (PG)-synthesis proteins suggests that the Chlamydiaceae possess the ability to synthesize PG yet biochemical evidence for the synthesis of PG has yet to be demonstrated. The presence of D-amino acids in PG is a hallmark of bacteria. Chlamydiaceae do not appear to encode amino acid racemases however, a D-alanyl-D-alanine (D-Ala-D-Ala) ligase homologue (Ddl) is encoded in the genome. Thus, we undertook a genetics-based approach to demonstrate and characterize the D-Ala-D-Ala ligase activity of chlamydial Ddl, a protein encoded as a fusion with MurC. The full-length murC-ddl fusion gene from Chlamydia trachomatis serovar L2 was cloned and placed under the control of the arabinose-inducible ara promoter and transformed into a D-Ala-D-Ala ligase auxotroph of Escherichia coli possessing deletions of both the ddlA and ddlB genes. Viability of the E. coliDeltaddlADeltaddlB mutant in the absence of exogenous D-Ala-D-Ala dipeptide became dependent on the expression of the chlamydial murC-ddl thus demonstrating functional ligase activity. Domain mapping of the full-length fusion protein and site-directed mutagenesis of the MurC domain revealed that the structure of the full fusion protein but not MurC enzymatic activity was required for ligase activity in vivo. Recombinant MurC-Ddl exhibited substrate specificity for D-Ala. Chlamydia growth is inhibited by D-cycloserine (DCS) and in vitro analysis provided evidence for the chlamydial MurC-Ddl as the target for DCS sensitivity. In vivo sensitivity to DCS could be reversed by addition of exogenous D-Ala and D-Ala-D-Ala. Together, these findings further support our hypothesis that PG is synthesized by members of the Chlamydiaceae family and suggest that D-amino acids, specifically D-Ala, are present in chlamydial PG.

Amino Acid Sequence↗
PubMed2005

Assays for RING family ubiquitin ligases.

Many eukaryotic proteins are regulated by the covalent attachment of ubiquitin or polyubiquitin chains. These include proteins involved in cell cycle control, tumor suppression, and many signaling pathways. Ubiquitination of proteins occurs through an enzymatic cascade of three discrete steps, which results in covalent attachment of ubiquitin to the substrate. The first two steps in this cascade involve the activating and conjugating enzymes, E1 and E2. The third and final step is performed by the E3 ubiquitin ligase. The ubiquitin ligase is responsible for two distinct activities: targeting specific substrates by bringing the substrate and activated ubiquitin together, as well as catalyzing the ligation of ubiquitin to the substrate. There are two main classes of E3 ligases, the HECT domain and the RING finger-containing ligases. RING finger-based ubiquitination utilizes RING-containing protein subunits, or proteins with intrinsic RING domains, to catalyze the formation of polyubiquitin chains. In this chapter we describe step by step protocols to assay for the activity of the RING finger-type of E3 ligase both in vitro and in vivo.

Amino Acid Motifs↗
PubMedDate not supplied

Mdm2 ligase dead mutants did not act in a dominant negative manner to re-activate p53, but promoted tumor cell growth.

Mdm2 (murine double minute 2) is an oncogene, first identified in BALB/c 3T3 cells. Over-expression and gene amplification of Mdm2 were found in a variety of human cancers. Recently, Mdm2 was found to be an E3 ubiquitin ligase that promotes degradation of p53, which contributes significantly to its oncogenic activity. In this study, we test a hypothesis that Mdm2 ligase dead mutants, which retained p53 binding activity but lost degradation activity, would act in a dominant negative manner to re-activate p53, especially upon stressed conditions. Five Mdm2 constructs expressing wild-type and E3 ligase-dead Mdm2 proteins were generated in a Tet-Off system and transfected into MCF-7 breast cancer cells (p53+/+ with Mdm2 overexpression) as well as MCF10A immortalized breast cells (p53+/+ without Mdm2 overexpression) as a normal control. We found that expression of Mdm2 mutants were tightly regulated by doxycycline. Withdrawal of doxycycline in culture medium triggered overexpression of Mdm2 mutants. However, expression of ligase dead mutants in MCF7 and MCF10A cells did not reactivate p53 as shown by a luciferase-reporter transcription assay and Western blot of p53 and its downstream target p21 under either unstressed condition or after exposure to DNA damaging agents. Biologically, over-expression of Mdm2 mutants had no effect on p53-induced apoptosis following DNA damage. Interestingly, over-expression of Mdm2 mutants promoted growth of MCF7 tumor cells probably via a p53-independent mechanism. Over-expression of Mdm2 mutants, however, had no effect on the growth of normal MCF10A cells and did not cause their transformation. Thus, ligase dead mutants of Mdm2 did not act in a dominant negative manner to reactivate p53 and they are not oncogenes in MCF10A cells.

Apoptosis↗
PubMed1987

Bacteriophage T4 anticodon nuclease, polynucleotide kinase and RNA ligase reprocess the host lysine tRNA.

Host tRNAs cleaved near the anticodon occur specifically in T4-infected Escherichia coli prr strains which restrict polynucleotide kinase (pnk) or RNA ligase (rli) phage mutants. The cleavage products are transient with wt but accumulate in pnk- or rli- infections, implicating the affected enzymes in repair of the damaged tRNAs. Their roles in the pathway were elucidated by comparing the mutant infection intermediates with intact tRNA counterparts before or late in wt infection. Thus, the T4-induced anticodon nuclease cleaves lysine tRNA 5' to the wobble position, yielding 2':3'-P greater than and 5'-OH termini. Polynucleotide kinase converts them into a 3'-OH and 5' P pair joined in turn by RNA ligase. Presumably, lysine tRNA depletion, in the absence of polynucleotide kinase and RNA ligase mediated repair, underlies prr restriction. However, the nuclease, kinase and ligase may benefit T4 directly, by adapting levels or decoding specificities of host tRNAs to T4 codon usage.

Base Sequence↗
PubMed2003

Isolation, sequencing, and expression of a cDNA for the HXM-A form of xenobiotic/medium-chain fatty acid:CoA ligase from human liver mitochondria.

The purification of xenobiotic/medium-chain fatty acid:CoA ligases (XM-ligases) from human liver mitochondria resulted in the isolation of two chromatographically separable forms (HXM-A and HXM-B). These two forms were purified to near homogeneity, cleaved with cyanogen bromide, the resulting peptides separated, and the N-terminus of two of the peptides partially sequenced. Identical sequences were obtained for HXM-A and HXM-B for the two peptides. These sequences were used to design probes for screening a human liver cDNA library. This resulted in the isolation of two overlapping cDNAs. Using these sequences we were able to design PCR primers that resulted in the isolation of a full-length cDNA from a human cDNA library. The cDNA contained 1731 bp of open reading frame and coded for a 64230-Da protein. This protein bears 56.2% amino acid homology to the MACS1 (medium-chain acyl-CoA synthetase) enzyme, 58.7% homology to the bovine XL-III XM-ligase, and 81.5% homology to the bovine XL-I XM-ligase. The cDNA could be expressed in COS cells, and the expressed enzyme had greater benzoate activity than phenylacetate activity, which is consistent with the known substrate specificity of HXM-A.

Amino Acid Sequence↗
PubMed1998

Cloning, expression, and chromosomal localization of human long-chain fatty acid-CoA ligase 4 (FACL4).

Long-chain fatty acid-CoA ligase (also called fatty acid acyl-CoA synthetase) plays an essential role in lipid biosynthesis and fatty acid degradation. We report herein the cDNA cloning of the human long-chain fatty acid-CoA ligase 4 from a brain library. The cDNA encodes a functional long-chain fatty acid-CoA ligase that shows preference for arachidonic acid as substrate. We also studied the tissue distribution of gene expression by Northern hybridization. Human placenta, brain, testis, ovary, spleen, and adrenal cortex have the highest levels of expression of the long-chain fatty acid-CoA ligase 4, whereas the GI system has the lowest. Finally, this gene was localized to chromosome Xq23 in human by FISH analysis.

Amino Acid Sequence↗
PubMed1986

Nucleotide sequence of the lig gene and primary structure of DNA ligase of Escherichia coli.

The DNA ligase of Escherichia coli catalyses the NAD-dependent formation of phosphodiester linkages between 5'-phosphoryl and 3'-hydroxyl groups in DNA. It is essential for DNA replication and repair of damaged DNA strands. We determined the nucleotide sequence of the lig gene of Escherichia coli coding for DNA ligase and flanking regions. The coding frame of the gene was confirmed by the amino acid composition and the amino- and carboxyl-terminal amino acid sequences of the purified ligase. The ligase consists of 671 amino acid residues with a molecular weight of 73,690.

Amino Acid Sequence↗
PubMed1989

Suppression of the thermosensitive DNA ligase mutations in Escherichia coli K12 through modulation of gene expression induced by phage Mu.

We have previously shown that Mu can sustain the growth at non-permissive temperature of an Escherichia coli strain harbouring a thermosensitive mutation in the DNA ligase structural gene. This "complementation" reaches a maximal level with the Mu lig3 mutant which restores the viability of a ligts7 strain to the level of the wild type (Ghelardini et al. 1980; Paolozzi et al. 1980). In this study we analysed the characteristics of this phenotypic suppression in order to clarify its molecular mechanism. We found that an E. coli ligts7 strain lysogenic for the Mu lig3 mutant shows: (i) an increment in the host DNA ligase activity; (ii) an increase in the specific mRNA of the host lig gene; (iii) an increase (towards the relaxed state) in the average linking number of a resident plasmid; and (iv) a reduction in DNA gyrase activity. These results are compatible with the hypothesis that the Mu lig gene product by interfering with the host enzymatic apparatus controlling DNA topology leads to a reduction in chromosomal supercoiling. The relaxation of the chromosome could affect the transcription of the DNA ligase gene, amongst others. Thus, through this mechanism, the Mu lig gene product is able to modulate gene expression and hence suppress the effects of the E. coli ligts7 mutation. On the basis of the identification of this mechanism of action, we propose to change the name of the Mu lig gene (thought originally to be the structural gene for a bacteriophage ligase) to gem (gene expression modulation).

Bacteriophage mu↗
PubMed1995

Molecular cloning and sequencing of human palmitoyl-CoA ligase and its tissue specific expression.

A complimentary DNA clone encoding the entire human palmitoyl-CoA ligase has been isolated from a liver cDNA library and sequenced in it's entirety. The predicted product is a 699 amino acid protein. Southern analysis utilizing the human palmitoyl-CoA ligase gene as a probe revealed varying degrees of similarity amongst various mammalian species. The palmitoyl-CoA ligase gene is highly expressed in liver, heart, skeletal muscle and kidney, and to a lesser extent in brain, lung, placenta and pancreas. The expression of palmitoyl-CoA ligase in various tissue parallels the function of this enzyme in the metabolism of fatty acids in these tissues.

Amino Acid Sequence↗
PubMed1987

Biosynthesis of glycerolipids by hepatoma and liver microsomes. I. Fatty acyl-CoA ligase and acyl-CoA:sn-glycerol-3-phosphate acyltransferase.

The intracellular membranes of hepatomas exhibit an altered content and composition of lipid compared to the membranes of normal liver. In order to elucidate the role of lipid biosynthetic enzymes in these membrane differences, we first examined the fatty acyl-CoA ligase and acyl-CoA:sn-glycerol 3-phosphate (Gro-3P) acyltransferase activities and acyl specificities of microsomes from liver, Morris hepatoma 7288C, and hepatoma tissue culture (HTC) cells. Based upon incorporation of fatty acid and Gro-3P, it is concluded that acyl-CoA:sn-Gro-3P acyltransferase activities are markedly elevated (6-30-fold) in the microsomes of Morris Hepatoma 7288C and HTC cells compared to microsomes from liver, whereas the fatty acyl-CoA ligase activity is reduced (30-50-fold). Therefore, the low phospholipid content of these tumor cells does not appear to result from reduced acyltransferase activity. Though diminished ligase activity may play a role, it appears that activation of fatty acid may not be rate-limiting, even at the low levels of fatty acyl-CoA ligase present in the tumor and HTC cells. Preliminary evidence suggests that another factor that may be responsible for the low tumor phospholipid content is the limited availability of Gro-3P, a lipid precursor. The phospholipid in hepatoma 7288C is also characterized by an elevated ratio of monenoic to dienoic fatty acid. We have found that this change does not reflect an altered specificity of acyl-CoA:sn-Gro-3P acyltransferase.

Acyltransferases↗
PubMed1979

Evidence for a single species of DNA ligase localized in nuclei of rat liver.

DNA ligase activity was distributed mainly in the extract of nuclei rapidly isolated from rat liver and about 20% of the nuclear activity in the cytoplasmic fraction. The DNA ligases of nuclear extract and cytoplasmic fraction showed similar gel-filtration patterns and the same sedimentation coefficient of 5.5 S. The appearance of 4-S enzyme from nuclei or nuclear extract was partly inhibited by phenylmethylsulfonyl fluoride. These results suggest that the major activity of DNA ligase in rat liver is due to a single species of the enzyme localized in nuclei and that 4-S nuclear DNA ligase reported previously is an artifact arising from 5.5-S enzyme during the isolation procedure.

Animals↗
PubMed1995

Kinetic characteristics of rat liver peroxisomal nafenopin-CoA ligase.

In this study we have demonstrated that rat hepatic peroxisomes catalyse the formation of nafenopin-CoA. The process is mediated by apparent high affinity (Km 6.7 microM), low capacity (Vmax 0.31 nmol/mg/min) and low affinity, high capacity isoforms. Palmitic acid (Ki 1.1 microM), R(-) ibuprofen (Ki 7.9 microM), ciprofibrate (Ki 60.2 microM) and clofibric acid (Ki 86.8 microM) competitively inhibited nafenopin-CoA formation catalysed by the apparent high affinity isoform. An antibody raised against the microsomal palmitoyl-CoA ligase inhibited the equivalent peroxisomal enzyme significantly (P < 0.001) but did not inhibit peroxisomal nafenopin-CoA ligase activity. These data suggest that nafenopin-CoA formation is catalysed by a peroxisomal CoA ligase which differs from the peroxisomal long chain fatty acid-CoA ligase in relation to its xenobiotic/antibody inhibitor profile and kinetic characteristics.

Animals↗