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At least 523 records · Page 29Linked to original sources
PubMed2005

Degradation of the tumor suppressor Smad4 by WW and HECT domain ubiquitin ligases.

Smad4 mediates signaling by the transforming growth factor-beta (TGF-beta) superfamily of cytokines. Smad signaling is negatively regulated by inhibitory (I) Smads and ubiquitin-mediated processes. Known mechanisms of proteasomal degradation of Smads depend on the direct interaction of specific E3 ligases with Smads. Alternatively, I-Smads elicit degradation of the TGF-beta receptor by recruiting the WW and HECT domain E3 ligases, Smurfs, WWP1, or NEDD4-2. We describe an equivalent mechanism of degradation of Smad4 by the above E3 ligases, via formation of ternary complexes between Smad4 and Smurfs, mediated by R-Smads (Smad2) or I-Smads (Smad6/7), acting as adaptors. Smurfs, which otherwise cannot directly bind to Smad4, mediated poly-ubiquitination of Smad4 in the presence of Smad6 or Smad7. Smad4 co-localized with Smad7 and Smurf1 primarily in the cytoplasm and in peripheral cell protrusions. Smad2 or Smad7 mutants defective in Smad4 interaction failed to induce Smurf1-mediated down-regulation of Smad4. A Smad4 mutant defective in Smad2 or Smad7 interaction could not be effectively down-regulated by Smurf1. We propose that Smad4 is targeted for degradation by multiple ubiquitin ligases that can simultaneously act on R-Smads and signaling receptors. Such mechanisms of down-regulation of TGF-beta signaling may be critical for proper physiological response to this pathway.

Adenoviridae↗
PubMed2001

APC2 Cullin protein and APC11 RING protein comprise the minimal ubiquitin ligase module of the anaphase-promoting complex.

In mitosis, the anaphase-promoting complex (APC) regulates the onset of sister-chromatid separation and exit from mitosis by mediating the ubiquitination and degradation of the securin protein and mitotic cyclins. With the use of a baculoviral expression system, we have reconstituted the ubiquitin ligase activity of human APC. In combination with Ubc4 or UbcH10, a heterodimeric complex of APC2 and APC11 is sufficient to catalyze the ubiquitination of human securin and cyclin B1. However, the minimal APC2/11 ubiquitin ligase module does not possess substrate specificity, because it also ubiquitinates the destruction box deletion mutants of securin and cyclin B1. Both APC11 and UbcH10 bind to the C-terminal cullin homology domain of APC2, whereas Ubc4 interacts with APC11 directly. Zn(2+)-binding and mutagenesis experiments indicate that APC11 binds Zn(2+) at a 1:3 M ratio. Unlike the two Zn(2+) ions of the canonical RING-finger motif, the third Zn(2+) ion of APC11 is not essential for its ligase activity. Surprisingly, with Ubc4 as the E2 enzyme, Zn(2+) ions alone are sufficient to catalyze the ubiquitination of cyclin B1. Therefore, the Zn(2+) ions of the RING finger family of ubiquitin ligases may be directly involved in catalysis.

Amino Acid Sequence↗
PubMed2001

Etoposide induces the dispersal of DNA ligase I from replication factories.

In eukaryotic cells DNA replication occurs in specific nuclear compartments, called replication factories, that undergo complex rearrangements during S-phase. The molecular mechanisms underlying the dynamics of replication factories are still poorly defined. Here we show that etoposide, an anticancer drug that induces double-strand breaks, triggers the redistribution of DNA ligase I and proliferating cell nuclear antigen from replicative patterns and the ensuing dephosphorylation of DNA ligase I. Moreover, etoposide triggers the formation of RPA foci, distinct from replication factories. The effect of etoposide on DNA ligase I localization is prevented by aphidicolin, an inhibitor of DNA replication, and by staurosporine, a protein kinase inhibitor and checkpoints' abrogator. We suggest that dispersal of DNA ligase I is triggered by an intra-S-phase checkpoint activated when replicative forks meet topoisomerase II-DNA--cleavable complexes. However, etoposide treatment of ataxia telangiectasia cells demonstrated that ataxia-telangiectasia-mutated activity is not required for the disassembly of replication factories and the formation of replication protein A foci.

Antineoplastic Agents, Phytogenic↗
PubMed1988

Peplomycin. DNA breakage and in vivo inhibition of DNA polymerases and ligase from human normal and leukemic cells.

Peplomycin, a bleomycin-related cytostatic agent, was tested on DNA polymerases and DNA ligase. These enzymes were purified from normal human immunocompetent cells (thymocytes and lymphocytes) and from peripheral blast cells from different kinds of acute lymphoblastic and acute non-lymphoblastic leukemia. At low concentration ranges (1-25 microM) this compound was found to strongly inhibit polymerase alpha and ligase from leukemic cells while being less effective on the enzyme activity from normal thymocytes and lymphocytes. At the DNA level, low concentrations of peplomycin resulted in the induction of dose-dependent single-stranded breaks. The incubation of peplomycin (5 microM) with plasmid DNA resulted in its degradation as observed by agarose gel electrophoresis. Lowering the peplomycin concentration showed that ligase inhibition takes place prior to this phenomenon. The decreased formation of the ligase--adenylate complex under the effect of peplomycin is consistent with a direct interaction between the drug and the enzyme. These results are discussed in terms of possible selective cytostatic effects of peplomycin on leukemic cells.

Bleomycin↗
PubMed1998

DNA ligase I is recruited to sites of DNA replication by an interaction with proliferating cell nuclear antigen: identification of a common targeting mechanism for the assembly of replication factories.

In mammalian cells, DNA replication occurs at discrete nuclear sites termed replication factories. Here we demonstrate that DNA ligase I and the large subunit of replication factor C (RF-C p140) have a homologous sequence of approximately 20 amino acids at their N-termini that functions as a replication factory targeting sequence (RFTS). This motif consists of two boxes: box 1 contains the sequence IxxFF whereas box 2 is rich in positively charged residues. N-terminal fragments of DNA ligase I and the RF-C large subunit that contain the RFTS both interact with proliferating cell nuclear antigen (PCNA) in vitro. Moreover, the RFTS of DNA ligase I and of the RF-C large subunit is necessary and sufficient for the interaction with PCNA. Both subnuclear targeting and PCNA binding by the DNA ligase I RFTS are abolished by replacement of the adjacent phenylalanine residues within box 1. Since sequences similar to the RFTS/PCNA-binding motif have been identified in other DNA replication enzymes and in p21(CIP1/WAF1), we propose that, in addition to functioning as a DNA polymerase processivity factor, PCNA plays a central role in the recruitment and stable association of DNA replication proteins at replication factories.

Amino Acid Sequence↗
PubMed2000

Crystal structure of NAD(+)-dependent DNA ligase: modular architecture and functional implications.

DNA ligases catalyze the crucial step of joining the breaks in duplex DNA during DNA replication, repair and recombination, utilizing either ATP or NAD(+) as a cofactor. Despite the difference in cofactor specificity and limited overall sequence similarity, the two classes of DNA ligase share basically the same catalytic mechanism. In this study, the crystal structure of an NAD(+)-dependent DNA ligase from Thermus filiformis, a 667 residue multidomain protein, has been determined by the multiwavelength anomalous diffraction (MAD) method. It reveals highly modular architecture and a unique circular arrangement of its four distinct domains. It also provides clues for protein flexibility and DNA-binding sites. A model for the multidomain ligase action involving large conformational changes is proposed.

Amino Acid Sequence↗
PubMed1982

Increased activity of polynucleotide ligase in 5-iodo-2'-deoxyuridine and mitomycin C-pretreated simian virus 40 (SV40)-infected monkey kidney cells.

The DNA ligase activity has been studied in 5-iodo-2'-deoxyuridine (IUdR), mitomycin C (MMC) and IUdR + MMC-treated SV40-infected or mock-infected CV1C11 monkey kidney cells. The results have shown that : 1. The level of enzyme activity is about two or three times higher in IUdR or MMC-treated non-infected cells; 2. SV40 infection doubles the level of ligase activity in the untreated cells; 3. There is an additional increase of ligase activity in IUdR or MMC-treated SV40-infected cells; 4. When infected or mock-infected cells are treated with IUdR + MMC, there is no modification of the results obtained with each drug alone; 5. Two peaks of different S values are detected in a partial purification of the drug(s) or viral induced enzyme(s). The increase in the ligase activity in drug-treated cells is independent of semi-conservative DNA synthesis. The drug(s) and viral-induced enzyme(s) is the consequence of a "de novo" synthesis of proteins, as shown by cycloheximide treatment.

Animals↗
PubMed1983

Isolation of the messenger RNA for 8S DNA ligase in early developing axolotl egg and its cell free translation.

A new DNA ligase activity is expressed when the Axolotl eggs enter cleavage. The messenger RNA can be labelled by [3H] uridine thereby indicating its de novo synthesis. This new genetic expression is occurring just before cleavage and is the earliest found during Amphibian development. The newly synthesized [3H] mRNA can be translated in vitro in the rabbit reticulocyte lysate system. The resulting product is a 160 K protein specifically immunoprecipitated with the antiserum directed against 8S DNA ligase. This in vitro translated polypeptide exhibits 8S DNA ligase activity specific of activated or fertilized eggs but does not display 6S DNA ligase activity of non activated eggs.

Ambystoma↗
PubMed1984

Polymer-stimulated ligation: enhanced ligation of oligo- and polynucleotides by T4 RNA ligase in polymer solutions.

The effects of macromolecular crowding were tested on several reactions catalyzed by T4 RNA ligase. The rate of cyclization of oligoriboadenylates was stimulated up to 10-fold by relatively high concentrations of several polymers (polyethylene glycol (PEG) 8000 or 20,000; bovine plasma albumin; Ficoll 70). In addition, higher concentrations of PEG 8000 or PEG 20,000 allowed the novel formation of large linear products from the oligoriboadenylates. Also stimulated by high concentrations of PEG 8000 were the rate at which T4 RNA ligase joined p(dT)10 to oligoriboadenylates and the rate at which the enzyme activated p(dT)n by transfer of an adenylyl moiety from ATP to the oligonucleotides. These results with T4 RNA ligase are compared to earlier studies on the effects of crowding on DNA ligases.

Chemical Phenomena↗
PubMed1985

The nucleotide sequence of the DNA ligase gene (CDC9) from Saccharomyces cerevisiae: a gene which is cell-cycle regulated and induced in response to DNA damage.

The CDC9 gene of Saccharomyces cerevisiae encodes a DNA ligase, and we have determined the nucleotide sequence of a 3.85 kb fragment of DNA which encompasses the convergently transcribed CDC9 and CDC36 genes. S1 nuclease mapping has revealed a major 5' end for the CDC9 mRNA, and one major and one minor site for 3' polyadenylation. These two sites lie within the C-terminal coding region of the CDC36 gene, implying that these two genes are transcribed from overlapping sequences. An interesting structural feature of the CDC9 gene is a series of 6 hexanucleotide repeats (ATGATT) which occur within the 650 bp immediately upstream from the site of transcription initiation. These repeat elements may be implicated in the cell division cycle regulated expression of CDC9. Comparison of the predicted amino acid sequence of the yeast DNA ligase (Mr 84,806) with the sequences of the T4 and T7 bacteriophage DNA ligases reveals little similarity except for a stretch of approximately 45 amino acids, comprising 3 short homologous segments. This region may represent an ATP-binding domain common to polynucleotide ligases.

Amino Acid Sequence↗
PubMed1988

DNA unwinding and inhibition of T4 DNA ligase by anthracyclines.

The ability to alter DNA tertiary structure of ten anthracycline derivatives whose antitumor potency is known was studied by an assay that makes use of nicked circular DNA and bacteriophage T4 DNA ligase. This assay allows the detection of tertiary structure alterations caused by DNA binding of both intercalating and non-intercalating drugs. The determination of these events can be obtained at different temperatures in the range of activity of DNA ligase. The results indicate that anthracyclines alter the DNA tertiary structure but this property does not correlate with their cytotoxic or antitumor activities. An additional interesting finding was that several anthracyclines inhibit T4 DNA ligase. The inhibition can be complete and is a cubic function of drug concentration. The inhibition of DNA ligase does not correlate with the ability of anthracyclines to alter the tertiary structure of DNA but is dependent from the presence of an amino group on the sugar ring.

Antibiotics, Antineoplastic↗
PubMed1989

DNA ligase activity in human cell lines from normal donors and Bloom's syndrome patients.

DNA ligase activity was studied in several untransformed or virus-transformed human cell lines from normal donors and from Bloom's syndrome (BS) patients. This proneness genetic disease is characterized by several cytological abnormalities and cancer proneness and, recently, some transformed cell lines from these patients were described to present a reduced activity of DNA ligase I. Results presented in this work indicate that: (i) the total DNA ligase activity in crude extract from untransformed or transformed cell lines from several BS patients was significantly higher than in control cells; (ii) the partial purification of the enzyme after gel filtration on fast protein liquid chromatography of crude extracts from lymphoblastoid BS cells showed that the enzyme activity was eluted in a major 180 kDa form in which activity was higher than in control cells; (iii) the activity gel analysis of these enzyme fractions revealed that DNA ligase of human cells was correlated to a major 130 kDa polypeptide and, in BS cells, the extent of the activity of this band was equal or higher than that in control untransformed or transformed cells.

Bloom Syndrome↗
PubMed2000

Ligation reaction specificities of an NAD(+)-dependent DNA ligase from the hyperthermophile Aquifex aeolicus.

An NAD(+)-dependent DNA ligase from the hyperthermophilic bacterium Aquifex aeolicus was cloned, expressed in Escherichia coli and purified to homogeneity. The enzyme is most active in slightly alkaline pH conditions with either Mg(2+)or Mn(2+)as the metal cofactor. Ca(2+)and Ni(2+)mainly support formation of DNA-adenylate intermediates. The catalytic cycle is characterized by a low k (cat)value of 2 min(-1)with concomitant accumulation of the DNA - adenylate intermediate when Mg(2+)is used as the metal cofactor. The ligation rates of matched substrates vary by up to 4-fold, but exhibit a general trend of T/A < or = G/C < C/G < A/T on both the 3'- and 5'-side of the nick. Consistent with previous studies on Thermus ligases, this Aquifex ligase exhibits greater discrimination against a mismatched base pair on the 3'-side of the nick junction. The requirement of 3' complementarity for a ligation reaction is reaffirmed by results from 1 nt insertions on either the 3'- or 5'-side of the nick. Furthermore, most of the unligatable 3' mismatched base pairs prohibit formation of the DNA-adenylate intermediate, indicating that the substrate adenylation step is also a control point for ligation fidelity. Unlike previously studied ATP ligases, gapped substrates cannot be ligated and intermediate accumulation is minimal, suggesting that complete elimination of base pair complementarity on one side of the nick affects substrate adenylation on the 5'-side of the nick junction. Relationships among metal cofactors, ligation products and intermediate, and ligation fidelity are discussed.

Base Pair Mismatch↗
PubMed1993

Cloning and gene map assignment of the Xiphophorus DNA ligase 1 gene.

Fishes represent the stem vertebrate condition and have maintained several gene arrangements common to mammalian genomes throughout the 450 Myr of divergence from a common ancestor. One such syntenic arrangement includes the GPI-PEPD enzyme association on Xiphophorus linkage group IV and human chromosome 19. Previously we assigned the Xiphophorus homologue of the human ERCC2 gene to linkage group U5 in tight association with the CKM locus. CKM is also tightly linked to the ERCC2 locus on human chromosome 19, leading to speculation that human chromosome 19 may have arisen by fusion of two ancestral linkage groups which have been maintained in fishes. To investigate this hypothesis further, we isolated and sequenced Xiphophorus fish genomic regions exhibiting considerable sequence similarity to the human DNA ligase 1 amino acid sequence. Comparison of the fish DNA ligase sequence with those of other species suggests several modes of amino acid conservation in this gene. A 2.2-kb restriction fragment containing part of an X. maculatus DNA ligase 1 exon was used in backcross hybrid mapping with 12 enzyme or RFLP loci. Significant linkage was observed between the nucleoside phosphorylase (NP2) and the DNA ligase (LIG1) loci on Xiphophorus linkage group VI. This assignment suggests that the association of four DNA repair-related genes on human chromosome 19 may be the result of chance chromosomal rearrangements.

Amino Acid Sequence↗
PubMed1980

Yeast mutants defective in acetyl-coenzyme A carboxylase and biotin: apocarboxylase ligase.

Among more than 7000 mutants of Saccharomyces cerevisiae, requiring saturated fatty acids, 61 acetyl-CoA-carboxylase-deficient strains have been identified. According to their mutual complementation characteristics these mutants have been assigned to two different genes, acc1 and acc2. Both acetyl-CoA carboxylase genes are unlinked to each other and to the fatty acids synthetase genes fas1 and fas2. The acetyl-CoA carboxylases of several acc1 and acc2 mutants have been purified and assayed for their overall and component enzyme activities. Besides overall acetyl-CoA carboxylation, which was lost in all cases, both component enzymes, biotin carboxylase and transcarboxylase, were simultaneously affected in most mutants, though often to a different relative extent. Similarly, the comparison of biochemical and genetic complementation data revealed no basis for a clear distinction between specific biotin carboxylase and transcarboxylase mutants. These results suggest that acc1 is a cluster gene coding for a multifunctional protein harboring both acetyl-CoA carboxylase component enzyme activities on the same polypeptide chain. The acetyl-CoA carboxylase isolated from acc2 mutants was free of biotin. Correspondingly, biotin:apoacetyl-CoA-carboxylase ligase activity was missing in acc2 mutants. Therefore, it is concluced that the primary defect in acc2 mutants is in the biotin:apocarboxylase ligase. In agreement with this conclusion, the acc2 acetyl-CoA carboxylase can be activated, in the presence of biotin and ATP, by ligase preparations from wild-type or acc1 mutant cells. By the use of these mutants, evidence was obtained that in vivo the biotinylation of both acetyl-CoA carboxylase and pyruvate carboxylase is catalyzed by the same ligase.

Acetyl-CoA Carboxylase↗
PubMed1982

Copurification of citrate lyase and citrate lyase ligase from Rhodopseudomonas gelatinosa and subsequent separation of the two enzymes.

A procedure has been worked out which allowed the purification and crystallization of a citrate lyase/citrate lyase ligase complex from Rhodopseudomonas gelatinosa. The complex was subsequently separated to yield two homogeneous enzymes. Citrate lyase ligase was purified 365-fold with a yield of 3.23%. The molecular weight of the enzyme was estimated to be 39500, the enzyme consisted of one polypeptide chain. The reaction rates for ATP, acetate and citrate lyase (sulfhydryl form) followed Michaelis-Menten kinetics (Km values: 0.14 mM, 5 mM and 37 nM respectively). Citrate lyase ligase exhibited a high substrate specificity and could not react with citrate lyases from nonphototrophic microorganisms. In contrast to the ligase from Streptococcus diacetilactis, the enzyme from R. gelatinosa was extremely labile; however, it could be stabilized by nucleotides, the most potent stabilizing one being ADP.

Carbon-Sulfur Ligases↗
PubMed1987

Mammalian DNA ligase. Structure and function in rat-liver tissues.

DNA ligase was partially purified from normal and regenerating rat liver. Its structure was studied using the activity gel procedure that identifies the functional polypeptides. Two slightly different purification procedures were followed leading to the isolation of one or two peaks (fractions A and B) of DNA ligase by hydroxyapatite chromatography. When analyzed on activity gels, all these enzyme fractions corresponded to a single active 130-kDa polypeptide both in normal and regenerating liver. A limited trypsin digestion of ligase fractions A and B gave rise to an identical pattern of smaller polypeptides of 110 kDa, 100 kDa and 75 kDa. Also storage at 4 degrees C of fractions A and B produced smaller polypeptides of 110 kDa, 100 kDa, 85 kDa and 60 kDa, which were identical for the two fractions. Our results indicate that the same ligase polypeptide of 130 kDa can be isolated from stationary or regenerating rat liver cells. However, physiological or artifactual proteolysis during various purification procedures can lead to the isolation of two enzyme fractions with different chromatographic behaviour but with the same molecular mass.

Animals↗
PubMed2025

Rice E3 ligase OsRFPH2-16 acts as a negative regulator to mediate the degradation of OsPIP1;1 under salt stress.

Soil salinity has a significant negative effect on rice productivity. We characterized the Oryza sativa RING Finger Protein H2-type-16 gene (OsRFPH2-16), which plays a negative role in response to salinity. The transcript levels of OsRFPH2-16 decreased under saline conditions. OsRFPH2-16 was expressed in the ER and tonoplasts of rice protoplasts. In addition, OsRFPH2-16 exhibited E3 ligase activity in an in vitro ubiquitination assay, whereas the mutant OsRFPH2-16C188A E3 ligase did not exhibit any activity. We constructed OsRFPH2-16-overexpressing (OX-2 and OX-4) and CRISPR/Cas9-mediated OsRFPH2-16-knockout (KO-4 and KO-16) plants and evaluated their salt responses. Under salt stress, OsRFPH2-16-knockout plants exhibited improved salt tolerance, characterized by low Na+ accumulation, high non-antioxidant content, and dynamic changes in the expression levels of Na+ transporter genes, compared with wild-type and OsRFPH2-16-overexpression plants. The aquaporin OsPIP1;1, an interacting partner, was identified using yeast two-hybridization, bimolecular fluorescence complementation, and pull-down assays. Degradation of OsPIP1;1 by the E3 ligase OsRFPH2-16 via the 26S proteasome system was confirmed through an in vitro degradation assay with the inhibitor MG132. These findings support that the E3 ligase functions as a negative regulator, leading to reduced Na+ accumulation in salt stress responses.

Oryza↗