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PubMed2002

Paramyxoviruses SV5 and HPIV2 assemble STAT protein ubiquitin ligase complexes from cellular components.

Signal transducer and activator of transcription (STAT) proteins are normally long-lived, but infection with certain Paramyxoviruses results in efficient loss of IFN-responsive STAT1 or STAT2. Expression of a virus-encoded protein called "V" is sufficient to mediate the destruction of STAT proteins. STAT degradation is blocked by proteasome inhibitors, strongly implicating the ubiquitin (Ub)-proteasome targeting system. We demonstrate that cellular expression of V proteins from simian virus 5 (SV5) and type II human parainfluenza virus (HPIV2) induces polyubiquitylation of STAT1 and STAT2 targets. In vitro, the V proteins catalyze Ub transfer in an ATP-dependent process that requires both Ub-activating (E1) and Ub-conjugating (E2) activities. Furthermore, SV5 and HPIV2 V-interacting protein partners were isolated by affinity purification from human cells and reveal a complex of associated cellular proteins. This complex includes both STAT1 and STAT2, and the damaged DNA binding protein, DDB1. In addition, a protein related to a family of cellular Ub ligase complex subunits, cullin 4A (Cul4A), associated with the V proteins. The roles of both DDB1 and Cul4A in STAT1 degradation by SV5 infection were analyzed using small interfering RNAs. These findings demonstrate the assembly of a V-dependent degradation complex that includes STAT1, STAT2, DDB1, and Cul4A. In agreement with prior nomenclature on SCF-type cellular E3 enzymes, we refer to this complex as VDC.

Antigen-Antibody Complex↗
PubMed1979

The DNA repair capability of cdc9, the Saccharomyces cerevisiae mutant defective in DNA ligase.

The cell cycle mutant, cdc9, in the yeast Saccharomyces cerevisiae is defective in DNA ligase be deficient in the repair of DNA damaged by methyl methane sulphonate. On the other hand survival of cdc9 after irradiation by gamma-rays is little different from that of the wild-type, even after a period of stress at the restrictive temperature. The mutant cdc9 is not allelic with any known rad or mms mutants.

DNA Ligases↗
PubMed1999

The pub1 E3 ubiquitin ligase negatively regulates leucine uptake in response to NH(4)(+) in fission yeast.

Fission yeast strains auxotrophic for leucine are unable to proliferate in normally supplemented minimal media adjusted to pH 6. 4 or above. High-pH sensitivity can be suppressed by the loss of Pub1, an E3 ubiquitin ligase, or by the replacement of NH(4)(+) with a non-repressing source of nitrogen such as L-proline. In this report we show pub1 to be required for the rapid down-regulation of leucine uptake observed in response to the addition of NH(4)(+) to the growth media. Furthermore, we corroborate earlier results demonstrating the transport of leucine to be negatively influenced by high extracellular pH. pub1 is homologous to the budding yeast nitrogen permease inactivator, NPI1/RSP5, which mediates the ubiquitination and subsequent destruction of NH(4)(+)-sensitive permeases. The high-pH sensitivity of cells auxotrophic for leucine thus seems to reflect an inability of NH(4)(+)-insensitive permeases to transport sufficient leucine under conditions where the proton gradient driving nutrient transport is low, and NH(4)(+)-sensitive permeases have been destroyed. Intriguingly, the partial suppression of both high pH sensitivity, and the inactivating effect of NH(4)(+) on leucine transport, seen in pub1-1 point mutants, becomes as complete as seen in pub1Delta backgrounds when cells have concomitantly lost the function of the spc1 stress-activated MAPK.

Cell Division↗
PubMed2006

UV-induced ubiquitylation of XPC complex, the UV-DDB-ubiquitin ligase complex, and DNA repair.

The DNA nucleotide excision repair (NER) system is our major defense against carcinogenesis. Defects in NER are associated with several human genetic disorders including xeroderma pigmentosum (XP), which is characterized by a marked predisposition to skin cancer. For initiation of the repair reaction at the genome-wide level, a complex containing one of the gene products involved in XP, the XPC protein, must bind to the damaged DNA site. The UV-damaged DNA-binding protein (UV-DDB), which is impaired in XP group E patients, has also been implicated in damage recognition in global genomic NER, but its precise functions and its relationship to the XPC complex have not been elucidated. However, the recent discovery of the association of UV-DDB with a cullin-based ubiquitin ligase has functionally linked the two damage recognition factors and shed light on novel mechanistic and regulatory aspects of global genomic NER. This article summarizes our current knowledge of the properties of the XPC complex and UV-DDB and discusses possible roles for ubiquitylation in the molecular mechanisms that underlie the efficient recognition and repair of DNA damage, particularly that induced by ultraviolet light irradiation, in preventing damage-induced mutagenesis as well as carcinogenesis.

DNA Damage↗
PubMed1979

The purification of nuclease-free T4-RNA ligase.

RNA ligase has been highly purified in good yields from bacteriophage T4-infected Escherichia coli by a rapid and reproducible procedure. The enzyme is free of phosphomonoesterase and ribonuclease activities and is therefore suitable for the synthesis of oligoribonucleotides and for the labeling of the 3'-terminus of RNA. Greater than 90% of the protein in the enzyme preparation migrates as a single band on gradient polyacrylamide gels containing sodium dodecyl sulfate during electrophoresis. For use as a DNA synthesis reagent the enzyme may be reliably freed of deoxyribonuclease activity by an additional chromatographic procedure using a commercially avialable resin.

Chromatography↗
PubMed1981

Efficiency of T4 DNA ligase-catalyzed end joining after S1 endonuclease treatment on duplex DNA containing single-stranded portions.

Covalently closed-circular, superhelical SV4O DNA was used in all experiments. EcoRI endonuclease- and HpaII endonuclease-generated unit-length linear duplex DNAs were digested with S1 endonuclease under the conditions where single-stranded CNA was completely converted into the acid-soluble form. These were subjected to an end-to-end joining test with T4 DNA ligase. The ligation efficiency was significantly lower than that of the flush-ended linear duplex DNAs which were generated by both HpaI endonuclease digestion and the matching up of EcoRI-generated sticky end with Escherichia coli DNA polymerase I (Klenow fraction). However, the ligation efficiency of the S1-treated DNAs increased up to same level as the flush-ended DNA upon treatment with E. coli DNA polymerase I . Similar results were obtained in the case of S1 -generated unit-length linear duplex DNA. S1 does cleave both strands of superhelical DNA at unbasepaired sites.

DNA Ligases↗
PubMed1985

Inhibition of DNA polymerase alpha, DNA polymerase beta, terminal deoxynucleotidyl transferase, and DNA ligase II by poly(ADP-ribosyl)ation reaction in vitro.

Incubation of DNA polymerase alpha, DNA polymerase beta, terminal deoxynucleotidyl transferase, or DNA ligase II in a reconstituted poly(ADP-ribosyl)ating enzyme system markedly suppressed the activity of these enzymes. Components required for poly(ADP-ribose) synthesis including poly(ADP-ribose) polymerase, NAD+, DNA, and Mg2+ were all essential for the observed suppression. Purified poly(ADP-ribose) itself, however, was slightly inhibitory to all of these enzymes. Furthermore, the suppressed activities of DNA polymerase alpha, DNA polymerase beta, and terminal deoxynucleotidyl transferase were largely restored (3 to 4-fold stimulation was observed) by a mild alkaline treatment, a procedure known to hydrolyze alkaline-labile ester linkage between poly(ADP-ribose) and an acceptor protein. All of these results strongly suggest that the four nuclear enzymes were inhibited as a result of poly(ADP-ribosyl)ation of either the enzyme molecule itself or some regulatory proteins of these enzymes.

Animals↗
PubMed1984

Reconstruction of tRNAPhe molecules from the fragments by linkage with T-4 RNA ligase in double-stranded regions.

Phenylalanine-specific tRNA from yeast was hydrolysed with cobra venom ribonuclease in the double-stranded regions and the fragments isolated. The 'dissected' molecules with nicks in positions 28 and 41 were reconstructed from supplementary fragments and treated with T-4 RNA ligase. A phosphodiester bond between two fragments was formed when the fragment combination (1-28) + (29-76) was used. A strong discrimination in the ligation yield between different nick positions in the same helix is shown.

Base Sequence↗
PubMed1993

Selective ubiquitination of calmodulin by UBC4 and a putative ubiquitin protein ligase (E3) from Saccharomyces cerevisiae.

A putative ubiquitin protein ligase (E3-CaM) which cooperates with UBC4 in selectively ubiquitinating calmodulin has been partially purified from Saccharomyces cerevisiae. Ca2+ was required for this activity and monoubiquitinated calmodulin was the main product of the reaction. The apparent Km of E3-CaM for calmodulin was approximately 1 microM which is of the same order of magnitude as the concentration of calmodulin in yeast cells. Proteins which are good substrates for other E3s (E3 alpha or E3-R) were not ubiquitinated by E3-CaM. Lower but significant activities of E3-CaM were observed when UBC1 replaced UBC4.

Calmodulin↗
PubMed1994

Purification and characterization of the VanB ligase associated with type B vancomycin resistance in Enterococcus faecalis V583.

Acquired resistance to glycopeptides in enterococci is associated with the production of D-Alanine:D-Alanine ligase-related proteins. The VanA protein associated with high-level vancomycin and teicoplanin resistance (VanA phenotype) synthesizes a new peptidoglycan precursor, D-alanine-D-lactate, that has reduced glycopeptide affinity. Production of a similar protein, VanB, is induced in strains that display variable levels of vancomycin resistance but remain susceptible to teicoplanin (VanB phenotype). This paper describes the over-production, purification and characterization of VanB. Comparison of kinetic parameters of the two Van enzymes suggests that differences in catalytic efficiency could account, at least in part, for the various levels of vancomycin resistance.

Amino Acid Sequence↗
PubMed1982

The use of a yeast strain with a temperature-sensitive DNA ligase to estimate DNA repair after exposure to mutagens.

The yeast strain cdc9 which possesses a temperature-sensitive DNA ligase, was used to estimate DNA repair after mutagen exposure. Following low UV fluences, single-strand breaks in DNA were detected after an incubation at the restrictive temperature but were absent at the permissive temperature. These DNA breaks were shown to be equal to the number of pyrimidine dimers induced in DNA as measured by the presence of UV-endonuclease sensitive sites. Similarly, after exposure to the chemical mutagen 4-chloromethyl-biphenyl (4CMB) single-strand breaks accumulated at the restrictive temperature. Hence the technique described should be applicable for the estimation of the early steps of repair of a wide range of different types of DNA damage induced in yeast by exposure to either physical or chemical mutagens.

DNA Ligases↗
PubMed1992

Use of long-chain fatty acid-CoA ligase (AMP-forming) from Pseudomonas fragi for the 'in vitro' synthesis of natural penicillins.

Five different naturally occurring penicillins containing as side chains hexanoic, trans-3-hexenoic, heptanoic, octanoic or trans-3-octenoic acids have been synthesized 'in vitro' by coupling long-chain fatty acid-CoA ligase (AMP-forming) (EC 6.2.1.3) from Pseudomonas fragi (LFCoA-L) with acyl-CoA: 6-aminopenicillanic acid acyltransferase (AT) from Penicillium chrysogenum. The quantity of penicillin produced was directly related with the carbon length of the side chain precursor tested, being maximal with octanoic acid. Fatty acids with a lower length than C5 were not recognized as substrates and nor were certain aromatic molecules.

Acyl Coenzyme A↗
PubMed1994

Cloning and sequence analysis of a cDNA coding for the murine DNA ligase I enzyme.

A complementary DNA (2961 bp) containing the complete coding sequence for murine DNA ligase I was isolated from a mouse fibroblast cDNA library using a cDNA encoding the human protein as a probe. An open reading frame of 2748 bp, encoding a protein of 916 amino acids (aa), was identified. Northern blot analysis of total RNA extracted from mouse fibroblasts showed a single band with a mobility corresponding to a size of 3.2 kb whose level increases upon serum stimulation of quiescent mouse NIH-3T3 cells. Alignment of the murine and human deduced aa sequences showed an overall 83% identity, that rises to 91% if only the sequence on the C-terminal portion of the protein containing the active site is considered.

3T3 Cells↗
PubMed1995

Comparison between cDNA clones encoding murine DNA ligase I.

A 3172-nucleotide (nt) cDNA clone encoding mouse DNA ligase I (LigI) was isolated from an embryonic stem cell cDNA library. Another mouse LigI cDNA clone has been recently described. Six single-amino-acid alterations have been identified between the two mouse LigI clones.

Animals↗
PubMed1996

The cloning and characterization of a cDNA encoding Xenopus laevis DNA ligase I.

A cDNA clone coding for DNA ligase I (LigI) was isolated from a Xenopus laevis oocyte cDNA library. The 3766-bp sequence showed a putative ORF capable of encoding a 1070-amino-acid protein whose overall identity with two mammalian sequences is 63%. This identity, however, rises to 72.5% in the C-terminal portion of the protein that contains the active site. Expression of the cDNA in a prokaryotic system produces a protein that is immunologically identical to LigI and can be adenylated. The 180-kDa size of the recombinant protein is similar to the LigI detected in oocyte. Northern blot analysis of ovary and embryo RNAs revealed the expression of two (4.1 and 6 kb) LigI transcripts.

Amino Acid Sequence↗
PubMed2004

Characterization of Rad6 from a higher plant, rice (Oryza sativa L.) and its interaction with Sgt1, a subunit of the SCF ubiquitin ligase complex.

We report here the existence of interactions between a ubiquitin-conjugating enzyme, Rad6, from rice, Oryza sativa L. cv. Nipponbare (OsRad6), and Sgt1 (OsSgt1), a novel subunit of the SCF ubiquitin ligase complex. Rad6 is not only related to post-replicational repair but also to the proteasome system, while Sgt1 has a function in kinetochore assembly. The relationship between the two is unexpected, but of great interest. The open reading frames of OsRad6 and OsSgt1 encode predicted products of 152 and 367 amino acid residues, respectively, with molecular weights of 17.3 and 40.9kDa. Two-hybrid and pull-down analyses indicated that OsRad6 binds to OsSgt1, and transcripts of both OsRad6 and OsSgt1 were found to be strongly expressed only in the proliferating tissues such as the shoot apical meristem, suggesting that their expression is cell cycle-dependent. The amount of the Rad6 mRNA in cultured cells increased rapidly after division was halted, and mRNA levels of Rad6 and Sgt1 were induced by UV- and DNA-damaging agents such as MMS or H(2)O(2). The Rad6 pathway for repair or the proteasome system may thus require Sgt1 as ubiquitin-conjugating enzyme.

Amino Acid Sequence↗
PubMed2004

The role of long-chain fatty-acid-CoA ligase 3 in vitamin D3 and androgen control of prostate cancer LNCaP cell growth.

The antiproliferative effect of 1alpha,25(OH)(2)D(3) on human prostate cancer cells is well known, but the mechanism is still not fully understood, especially its androgen-dependent action. Based on cDNA microarray results, we found that long-chain fatty-acid-CoA ligase 3 (FACL3/ACS3) might play an important role in vitamin D(3) and androgen regulation of LNCaP cell growth. The expression of FACL3/ACS3 was found to be significantly upregulated by 1alpha,25(OH)(2)D(3) and the regulation was shown to be time-dependent, with the maximal regulation over 3.5-fold at 96h. FACL3/ACS3 was a dominant isoform of FACL/ACS expressed in LNCaP cells as indicated by measuring the relative expression of each isoform. 1alpha,25(OH)(2)D(3) had no significant effect on the expression of FACL1(FACL2), FACL4 and FACL6 except for its downregulation of FACL5 at 24 and 48h by around twofold. The upregulation of FACL3/ACS3 expression by 1alpha,25(OH)(2)D(3) was accompanied with increased activity of FACL/ACS as demonstrated by enzyme activity assay using a (14)C-labeled substrate preferential for FACL3/ACS3. The growth inhibitory effect of 1alpha,25(OH)(2)D(3) on LNCaP cells was significantly attenuated by FACL3/ACS3 activity inhibitor. Androgen withdrawal (DCC-serum), in the presence of antiandrogen Casodex or in AR-negative prostate cancer cells (PC3 and DU145), vitamin D(3) failed to regulate FACL3/ACS3 expression. The upregulation of FACL3/ACS3 expression by vitamin D(3) was recovered by the addition of DHT in DCC-serum medium. Western blot analysis showed that the expression of androgen receptor (AR) protein was consistent with vitamin D(3) regulation of FACL3/ACS3 expression. Taken together, the data suggest that the upregulation of FACL3/ACS3 expression by vitamin D(3) is through an androgen/AR-mediated pathway and might be one of the contributions of the vitamin D(3) antiproliferative effect in prostate cancer LNCaP cells.

Androgens↗
PubMed2004

Ubiquitin-protein ligases in muscle wasting.

Muscle wasting occurs when rates of protein degradation outstrip rates of protein synthesis. Accelerated rates of protein degradation develop in atrophying muscle largely through activation of the ubiquitin-proteasome pathway. The complexity of the ubiquitination process, however, has hampered our understanding of how this pathway is activated in atrophying muscles and which enzymes of the ubiquitin conjugation system are responsible. Recent studies demonstrate that two ubiquitin-protein ligases (E3s), atrogin-1/MAFbx and MuRF1 are critical in the development of muscle atrophy. Other experiments implicate E2(14k) and E3alpha, of the N-end rule pathway, as important players in the process. It seems likely that multiple pathways of ubiquitin conjugation are activated in parallel in atrophying muscle, perhaps to target for degradation specific classes of muscle proteins. The emerging challenge will be to define the protein targets for, as well as to develop inhibitors of, these E3s.

Animals↗