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At least 955 records · Page 53Linked to original sources
PubMed1984

Quinolone antibiotics inhibit eucaryotic DNA polymerase alpha and beta, terminal deoxynucleotidyl transferase but not DNA ligase.

DNA polymerases alpha and beta, Terminal deoxynucleotidyl Transferase and DNA ligases from chicken thymus were purified to homogeneity. Quinolone antibiotics (nalidixic acid, oxolinic acid and pefloxacin ) known to inhibit DNA replication were tested for their effects on these enzymes. DNA ligase activity was not affected by the three drugs. DNA polymerases alpha and beta were inhibited by competitive mechanisms. Surprisingly, Terminal deoxynucleotidyl Transferase was strongly inhibited by the three compounds and more efficiently by nalidixic acid. The significance of these results is discussed in terms of the possible involvement of the enzymes in the respective DNA replication and repair processes.

Animals↗
PubMed1992

Inhibition kinetics of hepatic microsomal long chain fatty acid-CoA ligase by 2-arylpropionic acid non-steroidal anti-inflammatory drugs.

Microsomal long chain fatty acid CoA ligase (EC 6.2.1.3) has been implicated in the formation of CoA thioesters of xenobiotics containing a carboxylic acid moiety. In this study we have demonstrated that the microsomal enzyme from rat liver exhibits biphasic kinetics for the formation of palmitoyl-CoA, i.e. there are high affinity low capacity Kmhigh, 1.6 microM, Vmaxhigh, 12.9 nmol/mg/min) and low affinity high capacity (Kmlow, 506 microM, Vmaxlow, 58.3 nmol/mg/min) components. Inhibition of the high affinity isoform was studied using the R and S enantiomers of ibuprofen, fenoprofen, ketoprofen and naproxen. The high affinity component of palmitoyl-CoA formation was competitively inhibited by R-fenoprofen (Ki 15.4 microM) while R-ibuprofen exhibited mixed inhibition kinetics. In contrast the R and S enantiomers of ketoprofen and naproxen were non-competitive inhibitors. This diversity of inhibition kinetics observed argues in favour of a binding site in addition to the catalytic site. A competitive interaction with the high affinity form correlated with literature evidence of enantiospecific chiral inversion and "hybrid" triglyceride formation for the R enantiomers of fenoprofen and ibuprofen. Paradoxically, R-ketoprofen which is extensively inverted in rats was a non-competitive inhibitor of palmitoyl-CoA formation by the high affinity isoform suggesting that it may not act as an alternate substrate. The results of this study clearly indicate that formation of R-2-arylpropionate-CoAs is not fully explained by interaction with the high affinity isoform of a microsomal long chain (palmitoyl) CoA ligase and therefore the involvement of other isoforms cannot be discounted.

Animals↗
PubMed1987

Changes in the catalytic properties of DNA ligases during early sea urchin development.

Two distinct DNA ligases are expressed during early sea urchin embryogenesis. A light form (50 kDa) is found in unfertilized eggs (oocyte form) and a heavier enzyme (110 kDa) is observed at the two-cell stage (embryonic form). The chronology of the change reveals that the embryonic form is detected 90 min after fertilization. After the two proteins were purified, their catalytic properties were studied using different substrates. The oocyte ligase acts only on deoxypolymers while the embryonic form also ligates heteropolymers. The two enzymes were found to undergo both nick and cohesive-end ligation. With different kinds of restriction sites it was observed that the embryonic enzyme could also ligate blunt-ended DNA. These catalytic properties account for sealing of exogenous DNA and concatenation following DNA injection into eggs. The role of the oocyte form of the enzyme is unclear; one speculation is a role in repair of DNA breaks which might accumulate during long-term sperm and oocyte storage in the gonad.

Animals↗
PubMed1979

Evidence for a DNA ligase change related to early cleavage in axolotl egg.

A definite change in the forms of DNA ligase appears when the axolotl egg enters cleavage. Sucrose gradient and phosphocellulose chromatography show that the a 6S form of DNA ligase exists before division, i.e. in unfertilised and fertilised egg, and a 8.2S form is present at the first division. N-ethylmaleimide sensitivity and heat stability are different for the two forms. The possible significance of this early change is discussed.

Ambystoma↗
PubMed1993

The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53.

The ubiquitin-dependent proteolytic pathway plays a major role in selective protein degradation. Ubiquitination of proteins requires the sequential action of the ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzymes (E2), and in some cases ubiquitin-protein ligases (E3s). The oncogenic human papillomavirus (HPV) types 16 and 18 utilize this cellular proteolytic system to target the tumor suppressor protein p53. The HPV E6 oncoprotein binds to a cellular protein of 100 kd, termed E6-associated protein (E6-AP). The E6-E6-AP complex specifically interacts with p53, resulting in the rapid ubiquitin-dependent degradation of p53. Here we report the purification and identification of the factors necessary for the E6-E6-AP-mediated ubiquitination of p53. The ubiquitination of p53 requires the E1 enzyme and a novel E2 in mammalian cells, while E3 activity is conferred by the E6-E6-AP complex. Furthermore, E6-AP appears to have ubiquitin-protein ligase activity in the absence of E6.

Cell Line↗
PubMed1986

Cloning and characterization of the Schizosaccharomyces pombe DNA ligase gene CDC17.

The Schizosaccharomyces pombe CDC17 gene has been cloned by complementation of the cdc17 mutant coding for temperature-sensitive DNA ligase. An allele-specific suppressor active only in the presence of a high osmotic pressure was also isolated. The cloned CDC17 gene failed to complement the analogous DNA ligase mutation, cdc9, in Saccharomyces cerevisiae, although the reverse complementation was successful [Barker and Johnston, Eur. J. Biochem. 134 (1983) 315-319]. The CDC17 gene specifies a 2.8-kb transcript.

Cloning, Molecular↗
PubMed1992

Assignment of the gene encoding DNA ligase I to human chromosome 19q13.2-13.3.

The gene encoding DNA ligase I has been mapped on human chromosome 19 by analysis of rodent-human somatic cell hybrids informative for this chromosome and by two-color fluorescence in situ hybridization. The DNA ligase I gene (LIG1) is localized to 19q13.2-13.3 and is distal to ERCC1, the most telomeric of three DNA repair genes on this chromosome.

Blotting, Southern↗
PubMed2004

DNA polymerase lambda can elongate on DNA substrates mimicking non-homologous end joining and interact with XRCC4-ligase IV complex.

Non-homologous end joining (NHEJ) is one of two pathways responsible for the repair of double-strand breaks in eukaryotic cells. The mechanism involves the alignment of broken DNA ends with minimal homology, fill in of short gaps by DNA polymerase(s), and ligation by XRCC4-DNA ligase IV complex. The gap-filling polymerase has not yet been positively identified, but recent biochemical studies have implicated DNA polymerase lambda (pol lambda), a novel DNA polymerase that has been assigned to the pol X family, in this process. Here we demonstrate that purified pol lambda can efficiently catalyze gap-filling synthesis on DNA substrates mimicking NHEJ. By designing two truncated forms of pol lambda, we also show that the unique proline-rich region in pol lambda plays a role in limiting strand displacement synthesis, a feature that may help its participation in in vivo NHEJ. Moreover, pol lambda interacts with XRCC4-DNA ligase IV via its N-terminal BRCT domain and the interaction stimulates the DNA synthesis activity of pol lambda. Taken together, these data strongly support that pol lambda functions in DNA polymerization events during NHEJ.

Binding Sites↗
PubMed2005

The ubiquitin ligase SCF(Grr1) is required for Gal2p degradation in the yeast Saccharomyces cerevisiae.

F-box proteins represent the substrate-specificity determinants of the SCF ubiquitin ligase complex. We previously reported that the F-box protein Grr1p is one of the proteins involved in the transmission of glucose-generated signal for proteolysis of the galactose transporter Gal2p and fructose-1,6-bisphosphatase. In this study, we show that the other components of SCF(Grr1), including Skp1, Rbx1p, and the ubiquitin-conjugating enzyme Cdc34, are also necessary for glucose-induced Gal2p degradation. This suggests that transmission of the glucose signal involves an SCF(Grr1)-mediated ubiquitination step. However, almost superimposable ubiquitination patterns of Gal2p observed in wild-type and grr1Delta mutant cells imply that Gal2p is not the primary target of SCF(Grr1) ubiquitin ligase. In addition, we demonstrate here that glucose-induced Gal2p proteolysis is a cell-cycle-independent event.

Biodegradation, Environmental↗
PubMed2006

XLF interacts with the XRCC4-DNA ligase IV complex to promote DNA nonhomologous end-joining.

DNA nonhomologous end-joining (NHEJ) is a predominant pathway of DNA double-strand break repair in mammalian cells, and defects in it cause radiosensitivity at the cellular and whole-organism levels. Central to NHEJ is the protein complex containing DNA Ligase IV and XRCC4. By searching for additional XRCC4-interacting factors, we identified a previously uncharacterized 33 kDa protein, XRCC4-like factor (XLF, also named Cernunnos), that has weak sequence homology with XRCC4 and is predicted to display structural similarity to XRCC4. We show that XLF directly interacts with the XRCC4-Ligase IV complex in vitro and in vivo and that siRNA-mediated downregulation of XLF in human cell lines leads to radiosensitivity and impaired NHEJ. Furthermore, we establish that NHEJ-deficient 2BN cells derived from a radiosensitive and immune-deficient patient lack XLF due to an inactivating frameshift mutation in its gene, and that reintroduction of wild-type XLF into such cells corrects their radiosensitivity and NHEJ defects. XLF thus constitutes a novel core component of the mammalian NHEJ apparatus.

Amino Acid Sequence↗
PubMed2005

DNA ligase: getting a grip to seal the deal.

The crystal structure of human DNA ligase I catches the enzyme just before the last step of ligation and shows that the protein wraps completely around nicked DNA. The elegant structure explains how ligase attains fidelity for the sealing operation.

DNA Ligase ATP↗
PubMed1997

Purification and partial sequencing of the XL-I form of xenobiotic-metabolizing medium chain fatty acid:CoA ligase from bovine liver mitochondria, and its homology with the essential hypertension protein.

The XL-I form of xenobiotic-metabolizing medium-chain fatty acid:CoA ligase was purified to apparent homogeneity from bovine liver mitochondria. The procedure gave rise to a 435-fold increase in specific activity, with a yield of 12%. The enzyme eluted from a gel filtration column as a single peak with an apparent molecular weight of ca. 55,000. It ran as a single band on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) which had an apparent molecular weight of 62 kDa. N-Terminal sequence analysis of the enzyme gave no sequence, which indicates a blocked N-terminus. To obtain sequence data, the enzyme was cleaved at methionine residues using CNBr. The resulting peptides were separated by SDS-PAGE. The cleavage pattern revealed two large peptides with molecular weights of ca. 10,000 and 12,000, plus several smaller peptides of lesser intensity. The 10 kDa and 12 kDa peptides were electroblotted onto Trans-Blot, and then sequenced directly from the blot. The N-terminal sequences of these two peptides are presented. When compared with known sequences it was discovered that these two peptides both have high homology with regions of the SA essential hypertension protein. This suggests a role for a carboxylic acid:CoA ligase in the control of high blood pressure.

Amino Acid Sequence↗
PubMed2003

A novel mammalian endoplasmic reticulum ubiquitin ligase homologous to the yeast Hrd1.

The yeast hHrd1 is a ubiquitin-protein ligase (E3) involved in ER-associated degradation. It was originally identified by genetic methods as an E3 of the yeast cholesterol biosynthetic enzyme HMG-CoA reductase (HMGR). We report the identification and cloning of a human homologue of Hrd1 (hHrd1). Immunofluorescence imaging confirms that the endogenous hHrd1 resides in the ER and in vitro assay demonstrates that it has a ubiquitin-ligase activity. However, the homology between the human and yeast Hrd1 is limited to the N-terminal domain of the proteins, and hHrd1 does not appear to be involved in the degradation of mammalian HMGR.

Amino Acid Sequence↗
PubMed2000

Expression of fatty acid-CoA ligase 4 during development and in brain.

Fatty acid utilization is initiated by fatty acid-CoA ligase, which converts free fatty acids into fatty acyl-CoA esters. We have cloned previously the human long-chain fatty acid-CoA ligase 4 (FACL4), which is a central enzyme in controlling the free arachidonic acid level in cells and thereby regulating eicosanoid production. We report here the expression of this gene in tissues, particularly in different parts of the brain. We found that FACL4 encoded a 75 kDa enzyme and that there was a modified translation product expressed in the brain. FACL4 was expressed in early stages of development with a significant amount of FACL4 mRNA detected in an E7 mouse embryo. In addition, FACL4 was highly expressed in both adult and newborn mouse brain especially in the granule cells of the dentate gyrus and the pyramidal cell layer of CA1 in hippocampus, and the granular cell layer and Purkinje cells of the cerebellum.

Animals↗
PubMed2004

Transcriptional coregulator SNURF (RNF4) possesses ubiquitin E3 ligase activity.

SNURF/RNF4 has been implicated in transcriptional regulation and growth inhibition in a RING finger-dependent fashion. In this work, we show that SNURF mediates its own ubiquitination in vitro in a ubiquitin-conjugating enzyme (E2)-selective manner: SNURF acts as an E3 ligase with UbcH5A and B, HHR6B (RAD6B), E2-25K, MmUbc7 and UbcH13, but not with UbcH3, UbcM4, MmUbc6 or E2-20K. In contrast, the well-characterized RING E3, AO7, functions only with members of the UbcH5 family. Furthermore, depending on the E2 used, the ubiquitin modification manifests as mono- or multi-ubiquitination. Mutation of conserved cysteine residues within the RING finger motif of SNURF abolishes the ubiquitination in vitro and in intact cells. Size fractionation of murine embryonal carcinoma F9 cell proteins shows that the majority of endogenous SNURF resides in salt-resistant > or =500-kDa complexes, suggesting that SNURF functions as a RING component in a multiprotein complex. Taken together, SNURF/RNF4 functions as an E3 ligase and this activity is closely linked to its transcription regulatory functions.

Amino Acid Motifs↗
PubMed2005

Expression and assay of HECT domain ligases.

HECT domain ubiquitin ligases (HECT E3s), typified by human E6AP and yeast Rsp5p, are unique among the several classes of known ubiquitin ligases in that they participate directly in the chemistry of substrate ubiquitination reactions. This chapter discusses strategies for the expression of active HECT E3s and the assays that are available for analyzing E2 interaction, ubiquitin-thioester formation, and substrate ubiquitination.

Endosomal Sorting Complexes Required for Transport↗
PubMed2002

Polymorphism of DNA ligase I and risk of lung cancer--a case-control analysis.

DNA ligases catalyze the joining of single and double-strand DNA breaks, which is an essential step in DNA replication, recombination and repair. Recently, a common single nucleotide polymorphism (A-->C) in exon 6 of DNA ligase I (LIG1) was identified, but its functional relevance remains to be determined. Because LIG1 participates in DNA repair and reduced DNA repair capacity is associated with risk of lung cancer, we evaluated in a non-population-based case-control study of 530 lung cancer cases and 570 cancer-free controls the role of this polymorphism in susceptibility to lung cancer. All of the subjects were non-Hispanic whites and the controls were frequency-matched to cases on age, sex and smoking status. Using the polymerase chain reaction-restriction fragment length polymorphism method, we found that this LIGI A-->C substitution was very common in healthy controls and that the A and C allele frequencies were close to 0.5. However, there was no significant difference in the frequency distributions of LIGI genotypes between lung cancer cases and controls (25.7, 49.8 and 24.5% in cases and 26.1, 49.7 and 24.2% in controls for the AA, AC and CC genotypes, respectively). Therefore, there was no evidence to support an association between this polymorphism and the risk of lung cancer (adjusted odds ratio (OR)=1.06, 95% confidence interval (CI)=0.76-1.49 for AC versus CC and OR=0.93, 95% CI=0.64-1.36 for AA versus CC) neither in all cases nor in different histopathologic types. The results of this large case-control study suggest that this LIG1 polymorphism may not play an important role in susceptibility to lung cancer.

Adult↗
PubMed1983

Optimization of conditions for labeling the 3' OH end of tRNA using T4 RNA ligase.

For several years most primary structure studies of ribonucleic acids have used the [32P] in vitro post-labeling techniques. We adapted our methods from the literature, and simplified them to make them accessible to any laboratory. These procedures are especially useful for preparation and purification of post labeling enzymes: T4 polynucleotide kinase, T4 RNA ligase and of gamma [32P] ATP. We developed a test tube method for 5' [32P] pCp preparation followed by tRNA labeling with T4 RNA ligase. The parameters for optimal labeling were determined. Labeling of 3.10(6) to 5.10(6) Cerenkov CPM per microgram tRNA are currently obtained.

Adenosine Triphosphate↗