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At least 595 records · Page 33Linked to original sources
PubMed1995

T4 DNA ligase modulates chromosome damage induced by restriction endonucleases through an error-free process.

The possible modulation by T4 DNA ligase of the DNA double-strand breaks produced by restriction endonucleases in living mammalian cells was studied. A clear decrease in the frequency of chromosomal aberrations was observed when T4 DNA ligase was included in the electroporation treatment along with restriction enzymes inducing either cohesive- or blunt-ends in DNA. The relative proportions of exchange-type aberrations were similar for both kinds of treatments (with and without ligase), which seems to suggest an error-free ligation by T4 DNA ligase.

Animals↗
PubMed1995

Detection of point mutations with a modified ligase chain reaction (Gap-LCR).

DNA amplification systems are powerful technologies with the potential to impact a wide range of diagnostic applications. In this study we explored the feasibility and limitations of a modified ligase chain reaction (Gap-LCR) in detection and discrimination of DNAs that differ by a single base. LCR is a DNA amplification technology based on the ligation of two pairs of synthetic oligonucleotides which hybridize at adjacent positions to complementary strands of a target DNA. Multiple rounds of denaturation, annealing and ligation with a thermostable ligase result in the exponential amplification of the target DNA. A modification of LCR, Gap-LCR was developed to reduce the background generated by target-independent, blunt-end ligation. In Gap-LCR, DNA polymerase fills in a gap between annealed probes which are subsequently joined by DNA ligase. We have designed synthetic DNA targets with single base pair differences and analyzed them in a system where three common probes plus an allele-specific probe were used. A single base mismatch either at the ultimate 3' end or penultimate 3' end of the allele specific probe was sufficient for discrimination, though better discrimination was obtained with a mismatch at the penultimate 3' position. Comparison of Gap-LCR to allele-specific PCR (ASPCR) suggested that Gap-LCR has the advantage of having the additive effect of polymerase and ligase on specificity. As a model system, Gap-LCR was tested on a mutation in the reverse transcriptase gene of HIV, specifically, one of the mutations that confers AZT resistance. Mutant DNA could be detected and discriminated in the presence of up to 10,000-fold excess of wild-type DNA.

Base Sequence↗
PubMed1997

Domain structure of vaccinia DNA ligase.

The 552 amino acid vaccinia virus DNA ligase consists of three structural domains defined by partial proteolysis: (i) an amino-terminal 175 amino acid segment that is susceptible to digestion with chymotrypsin and trypsin; (ii) a protease-resistant central domain that contains the active site of nucleotidyl transfer (Lys-231); (iii) a protease-resistant carboxyl domain. The two protease-resistant domains are separated by a protease-sensitive interdomain bridge from positions 296 to 307. Adenylyltransferase and DNA ligation activities are preserved when the N-terminal 200 amino acids are deleted. However, the truncated form of vaccinia ligase has a reduced catalytic rate in strand joining and a lower affinity for DNA than does the full-sized enzyme. The 350 amino acid catalytic core of the vaccinia ligase is similar in size and protease-sensitivity to the full-length bacteriophage T7 DNA ligase.

Adenosine Triphosphate↗
PubMed1998

Chlorella virus DNA ligase: nick recognition and mutational analysis.

Chlorella virus PBCV-1 DNA ligase seals nicked DNA substrates consisting of a 5'-phosphate-terminated strand and a 3'-hydroxyl-terminated strand annealed to a bridging DNA template strand. The enzyme discriminates at the DNA binding step between substrates containing a 5'-phosphate versus a 5'-hydroxyl at the nick. Mutational analysis of the active site motif KxDGxR (residues 27-32) illuminates essential roles for the conserved Lys, Asp and Arg moieties at different steps of the ligase reaction. Mutant K27A is unable to form the covalent ligase-(Lys-straightepsilonN-P)-adenylate intermediate and hence cannot activate a nicked DNA substrate via formation of the DNA-adenylate intermediate. Nonetheless, K27A catalyzes phosphodiester bond formation at a pre-adenylated nick. This shows that the active site lysine is not required for the strand closure reaction. K27A binds to nicked DNA-adenylate, but not to a standard DNA nick. This suggests that occupancy of the AMP binding pocket of DNA ligase is important for nick recognition. Mutant D29A is active in enzyme-adenylate formation and binds readily to nicked DNA, but is inert in DNA-adenylate formation. R32A is unable to catalyze any of the three reactions of the ligation pathway and does not bind to nicked DNA.

Adenosine Monophosphate↗
PubMed2002

Role of nucleotidyltransferase motifs I, III and IV in the catalysis of phosphodiester bond formation by Chlorella virus DNA ligase.

ATP-dependent DNA ligases catalyze the sealing of 5'-phosphate and 3'-hydroxyl termini at DNA nicks by means of a series of three nucleotidyl transfer steps. Here we have analyzed by site-directed mutagenesis the roles of conserved amino acids of Chlorella virus DNA ligase during the third step of the ligation pathway, which entails reaction of the 3'-OH of the nick with the DNA-adenylate intermediate to form a phosphodiester and release AMP. We found that Asp65 and Glu67 in nucleotidyltransferase motif III and Glu161 in motif IV enhance the rate of step 3 phosphodiester formation by factors of 20, 1000 and 60, respectively. Asp29 and Arg32 in nucleotidyltransferase motif I enhance the rate of step 3 by 60-fold. Gel shift analysis showed that mutations of Arg32 and Asp65 suppressed ligase binding to a pre-adenylated nick, whereas Asp29, Glu67 and Glu161 mutants bound stably to DNA-adenylate. We infer that Asp29, Glu67 and Glu161 are involved directly in the step 3 reaction. In several cases, the effects of alanine or conservative mutations on step 3 were modest compared to their effects on the composite ligation reaction and individual upstream steps. These results, in concert with available crystallographic data, suggest that the active site of DNA ligase is remodeled during the three steps of the pathway and that some of the catalytic side chains play distinct roles at different stages.

Adenosine Monophosphate↗
PubMed1979

The effect of antibiotics on the T4 polynucleotide ligase catalyzed template dependent polymerization of oligodeoxythymidylates.

The poly(dA) dependent T4 polynucleotide ligase catalyzed polymerization of oligodeoxythymidylates is dependent upon duplex stability. The antibiotics ethidium bromide, netropsin and Hoechst 33258 stabilize the duplex poly(dA) . P(dT)n (n = 6-10) to thermal denaturation. Ethidium bromide to DNA ratio of 1.25 and netropsin or Hoechst 33258 to DNA ratio of 0.1 the Tm of d(pT) 10 . poly (dA) was increased by 10 degrees and 25 degrees C respectively. The T4 polynucleotide ligase activity was not inhibited under these conditions and temperature optimum of joining of d(pT) 10 . poly(dA) was increased 5 degrees to 10 degrees by the binding of the antibiotics. Duplexes containing shorter oligodeoxythymidylates required lower concentrations of the antibiotics netropsin or Hoechst 33258 to show no inhibition of T4 polynucleotide ligase. The temperature optima of joining the duplexes d(pT)6 . POLY(DA) and d(pT) 8 . poly(dA) were increased by 5 degrees C upon binding of the antibiotics. Polyacrylamide gel analysis of the T4 polynucleotide ligase catalyzed joining of the oligodeoxythymidylates showed that the presence of antibiotics affected the product distribution of the polymerized oligomers.

Anti-Bacterial Agents↗
PubMed2005

Isolation and characterization of a thermostable RNA ligase 1 from a Thermus scotoductus bacteriophage TS2126 with good single-stranded DNA ligation properties.

We have recently sequenced the genome of a novel thermophilic bacteriophage designated as TS2126 that infects the thermophilic eubacterium Thermus scotoductus. One of the annotated open reading frames (ORFs) shows homology to T4 RNA ligase 1, an enzyme of great importance in molecular biology, owing to its ability to ligate single-stranded nucleic acids. The ORF was cloned, and recombinant protein was expressed, purified and characterized. The recombinant enzyme ligates single-stranded nucleic acids in an ATP-dependent manner and is moderately thermostable. The recombinant enzyme exhibits extremely high activity and high ligation efficiency. It can be used for various molecular biology applications including RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE). The TS2126 RNA ligase catalyzed both inter- and intra-molecular single-stranded DNA ligation to >50% completion in a matter of hours at an elevated temperature, although favoring intra-molecular ligation on RNA and single-stranded DNA substrates. The properties of TS2126 RNA ligase 1 makes it very attractive for processes like adaptor ligation, and single-stranded solid phase gene synthesis.

Amino Acid Sequence↗
PubMed2004

Characterization of a temperature-sensitive DNA ligase from Escherichia coli.

DNA ligases are essential enzymes in cells due to their ability to join DNA strand breaks formed during DNA replication. Several temperature-sensitive mutant strains of Escherichia coli, including strain GR501, have been described which can be complemented by functional DNA ligases. Here, it is shown that the ligA251 mutation in E. coli GR501 strain is a cytosine to thymine transition at base 43, which results in a substitution of leucine by phenylalanine at residue 15. The protein product of this gene (LigA251) is accumulated to a similar level at permissive and non-permissive temperatures. Compared to wild-type LigA, at 20 degrees C purified LigA251 has 20-fold lower ligation activity in vitro, and its activity is reduced further at 42 degrees C, resulting in 60-fold lower ligation activity than wild-type LigA. It is proposed that the mutation in LigA251 affects the structure of the N-terminal region of LigA. The resulting decrease in DNA ligase activity at the non-permissive temperature is likely to occur as the result of a conformational change that reduces the rate of adenylation of the ligase.

Amino Acid Sequence↗
PubMed1999

The SCFbeta-TRCP-ubiquitin ligase complex associates specifically with phosphorylated destruction motifs in IkappaBalpha and beta-catenin and stimulates IkappaBalpha ubiquitination in vitro.

Ubiquitin-mediated proteolysis has a central role in controlling the intracellular levels of several important regulatory molecules such as cyclins, CKIs, p53, and IkappaBalpha. Many diverse proinflammatory signals lead to the specific phosphorylation and subsequent ubiquitin-mediated destruction of the NF-kappaB inhibitor protein IkappaBalpha. Substrate specificity in ubiquitination reactions is, in large part, mediated by the specific association of the E3-ubiquitin ligases with their substrates. One class of E3 ligases is defined by the recently described SCF complexes, the archetype of which was first described in budding yeast and contains Skp1, Cdc53, and the F-box protein Cdc4. These complexes recognize their substrates through modular F-box proteins in a phosphorylation-dependent manner. Here we describe a biochemical dissection of a novel mammalian SCF complex, SCFbeta-TRCP, that specifically recognizes a 19-amino-acid destruction motif in IkappaBalpha (residues 21-41) in a phosphorylation-dependent manner. This SCF complex also recognizes a conserved destruction motif in beta-catenin, a protein with levels also regulated by phosphorylation-dependent ubiquitination. Endogenous IkappaBalpha-ubiquitin ligase activity cofractionates with SCFbeta-TRCP. Furthermore, recombinant SCFbeta-TRCP assembled in mammalian cells contains phospho-IkappaBalpha-specific ubiquitin ligase activity. Our results suggest that an SCFbeta-TRCP complex functions in multiple transcriptional programs by activating the NF-kappaB pathway and inhibiting the beta-catenin pathway.

Amino Acid Sequence↗
PubMed2006

The E3 ubiquitin ligase activity of arabidopsis PLANT U-BOX17 and its functional tobacco homolog ACRE276 are required for cell death and defense.

Previous analysis of transcriptional changes after elicitation of Cf-9 transgenic tobacco (Nicotiana tabacum) by Avr9 peptide revealed a rapidly upregulated gene, ACRE276. We show that ACRE276 is transiently induced in wounded leaves within 15 min, but upon Avr9 elicitor treatment, this upregulation is enhanced and maintained until cell death onset in Cf-9 tobacco. ACRE276 RNA interference (RNAi) silencing in tobacco results in loss of hypersensitive response (HR) specified by Cf resistance genes. ACRE276 RNAi plants are also compromised for HR mediated by the tobacco mosaic virus defense elicitor p50. Silencing tomato (Lycopersicon esculentum) ACRE276 leads to breakdown of Cf-9-specified resistance against Cladosporium fulvum leaf mold. We confirmed that tobacco ACRE276 is an E3 ubiquitin ligase requiring an intact U-box domain. Bioinformatic analyses revealed Arabidopsis thaliana PLANT U-BOX17 (PUB17) and Brassica napus ARC1 as the closest homologs of tobacco ACRE276. Transiently expressing PUB17 in Cf-9 tobacco silenced for ACRE276 restores HR, while mutant PUB17 lacking E3 ligase activity fails to do so, demonstrating that PUB17 ligase activity is crucial for defense signaling. Arabidopsis PUB17 knockout plants are compromised in RPM1- and RPS4-mediated resistance against Pseudomonas syringae pv tomato containing avirulence genes AvrB and AvrRPS4, respectively. We identify a conserved class of U-box ARMADILLO repeat E3 ligases that are positive regulators of cell death and defense across the Solanaceae and Brassicaceae.

Amino Acid Sequence↗
PubMed2006

Arabidopsis CULLIN4 Forms an E3 Ubiquitin Ligase with RBX1 and the CDD Complex in Mediating Light Control of Development.

Repression of photomorphogenesis in Arabidopsis thaliana requires activity of the COP9 signalosome (CSN), CDD, and COP1 complexes, but how these three complexes work in concert to accomplish this important developmental switch has remained unknown. Here, we demonstrate that Arabidopsis CULLIN4 (CUL4) associates with the CDD complex and a common catalytic subunit to form an active E3 ubiquitin ligase both in vivo and in vitro. The partial loss of function of CUL4 resulted in a constitutive photomorphogenic phenotype with respect to morphogenesis and light-regulated gene expression. Furthermore, CUL4 exhibits a synergistic genetic interaction with COP10 and DET1. Therefore, this CUL4-based E3 ligase is essential for the repression of photomorphogenesis. This CUL4-based E3 ligase appears to associate physically with COP1 E3 ligase and positively regulates the COP1-dependent degradation of photomorphogenesis-promoting transcription factors, whereas the CSN controls the biochemical modification of CUL4 essential for E3 activity. Thus, this study suggests a biochemical activity connection between CSN and CDD complexes in their cooperation with COP1 in orchestrating the repression of photomorphogenesis.

Arabidopsis↗
PubMed2005

Overexpression, purification and crystallization of an archaeal DNA ligase from Pyrococcus furiosus.

DNA ligases seal single-strand breaks in double-stranded DNA and their function is essential to maintain the integrity of the genome during various aspects of DNA metabolism, such as replication, excision repair and recombination. DNA-strand breaks are frequently generated as reaction intermediates in these events and the sealing of these breaks depends solely on the proper function of DNA ligase. Crystals of the archaeal DNA ligase from Pyrococcus furiosus were obtained using 6.6%(v/v) ethanol as a precipitant and diffracted X-rays to 1.7 A resolution. They belong to the monoclinic space group P2(1), with unit-cell parameters a = 61.1, b = 88.3, c = 63.4 A, beta = 108.9 degrees. The asymmetric unit contains one ligase molecule.

Catalytic Domain↗
PubMed1995

RNA capping enzyme and DNA ligase: a superfamily of covalent nucleotidyl transferases.

mRNA capping entails GMP transfer from GTP to a 5' diphosphate RNA end to form the structure G(5')ppp(5')N. A similar reaction involving AMP transfer to the 5' monophosphate end of DNA or RNA occurs during strand joining by polynucleotide ligases. In both cases, nucleotidyl transfer occurs through a covalent lysyl-NMP intermediate. Sequence conservation among capping enzymes and ATP-dependent ligases in the vicinity of the active site lysine (KxDG) and at five other co-linear motifs suggests a common structural basis for covalent catalysis. Mutational studies support this view. We propose that the cellular and DNA virus capping enzymes and ATP-dependent ligases constitute a protein superfamily evolved from a common ancestral enzyme. Within this superfamily, the cellular capping enzymes display more extensive similarity to the ligases than they do to the poxvirus capping enzymes. Recent studies suggest that eukaryotic RNA viruses have evolved alternative pathways of cap metabolism catalysed by structurally unrelated enzymes that nonetheless employ a phosphoramidate intermediate. Comparative analysis of these enzymes, particularly at the structural level, should illuminate the shared reaction mechanism while clarifying the basis for nucleotide specificity and end recognition. The capping enzymes merit close attention as potential targets for antiviral therapy.

Amino Acid Sequence↗
PubMed2004

The ubiquitin ligase Rsp5p is required for modification and sorting of membrane proteins into multivesicular bodies.

Precursor forms of vacuolar proteins with transmembrane domains, such as the carboxypeptidase S Cps1p and the polyphosphatase Phm5p, are selectively sorted in endosomal compartments to vesicles that invaginate, budding into the lumen of the late endosomes, resulting in the formation of multivesicular bodies (MVBs). These proteins are then delivered to the vacuolar lumen following fusion of the MVBs with the vacuole. The sorting of Cps1p and Phm5p to these structures is mediated by ubiquitylation, and in doa4 mutant cells, which have reduced level of free ubiquitin, these proteins are missorted to the vacuolar membrane. A RING-finger ubiquitin ligase Tul1p has been shown to participate in the ubiquitylation of Cps1p and Phm5p. We show here that the HECT-ubiquitin ligase Rsp5p is also required for the ubiquitylation of these proteins, and therefore for their sorting to MVBs. Rsp5p is an essential ubiquitin ligase containing an N-terminal C2 domain followed by three WW domains, and a C-terminal catalytic HECT domain. In cells with low levels of Rsp5p (npi1 mutant cells), vacuolar hydrolases do not reach the vacuolar lumen and are instead missorted to the vacuolar membrane. The C2 domain and both the second and third WW domains of Rsp5p are important determinants for sorting to MVBs. Ubiquitylation of Cps1p was strongly reduced in the npi1 mutant strain and ubiquitylation was completely abolished in the npi1 tul1Delta double mutant. These data demonstrate that Rsp5p plays a novel and key role in intracellular trafficking, and extend the currently very short list of substrates ubiquitylated in vivo by several different ubiquitin ligases acting cooperatively.

Carboxypeptidases↗
PubMed1977

Purification and properties of citrate lyase ligase from Streptococcus diacetilactis.

Citrate lyase ligase (acetate: SH--[acyl-carrier protein] enzyme ligase (AMP) from Streptococcus diacetilactis was purified 920-fold with a yield of 6.3%. The molecular weight of the enzyme was estimated to be 41000; the ligase consisted of one polypeptide chain. The acetylation of 1 mol of deacetyl-citrate lyase to enzymatically active citrate lyase required 6 mol ATP. The formation of AMP and pyrophosphate in the acetylation reaction was demonstrated. Citrate lyase ligase was specific for the lyase from S. diacetilacitis and did not acetylate lyases from Rhodopseudomonas gelatinosa and Enterobacter aerogenes. The substract acetate and ATP could be replaced by propionate and dATP, repectively. The reaction rates for ATP, acetate and deacetyl-citrate lyase followed Michaelis-Menten kinetics (Km values: 26 micron for ATP, 25 mM for acetate and 38 nM for deacetyl-citrate lyase).

Citrates↗
PubMed1995

The gene encoding the biotin-apoprotein ligase of Saccharomyces cerevisiae.

We report the isolation, genomic mapping, and DNA sequence of the BPL1 gene encoding the biotin-apoprotein ligase of Saccharomyces cerevisiae. The gene was isolated by complementation of an Escherichia coli birA (biotin-apoprotein ligase) mutant indicating that the expressed yeast protein modified the essential biotinated protein of the bacterial host. The BPL1 gene encodes a protein of 690 residues (M(r) 76.4 kDa) with strong sequence similarities to the E. coli and human biotin-apoprotein ligases. BPL1 was mapped to chromosome IV, is allelic to the previously described ACC2 gene, and encodes the major (if not the only) biotin-apoprotein ligase activity of S. cerevisiae.

Alleles↗
PubMed1976

Role of deoxyribonucleic acid ligase in a doxyribonucleic acid membrane fraction extracted from pneumococci.

Deoxyribonucleic acid (DNA) ligase has been detected in a DNA membrane fraction extracted from Pneumococcus. The specific activity of the enzyme in this fraction is 10-fold greater than in the remaining cell extract. It remains firmly bound (with other enzymes) to the complex after a purification procedure in which a considerable percentage of the macromolecules are dissociated. The ligase acts in two ways in the DNA membrane fraction in vitro. One, it catalyzes the linkage of small-molecular-weight pieces of newly synthesized DNA into heavier-molecular-weight DNA strands as shown by others (M Gellert, 1976; R. Okazaki, A. Sugino, S. Hirose, T. Okazaki, Y. Imae, R. Kainuma-Kuroda, T. Ogawa, M. Arisawa, and Y. Kurosowa, 1973; B. Olivera and I. Lehman, 14; and A. Sugino, S. Hirose, and R. Okazaki, 1972) and, two, it protects DNA from degradation by deoxyribonucleases. This latter effect is due to a competition between the ability of the nucleases to degrade DNA and the ability of DNA ligase to seal the nicks produced by these degradative enzymes. The ligase acts cooperatively with other enzymes in the DNA membrane fraction to synthesize DNA.

Adenosine Triphosphate↗
PubMed1974

Transfection of Escherichia coli spheroplasts. IV. Transfection of rec+ and rec minus spheroplasts by native, denatured, and renatured T5 bacteriophage DNA after repair of single-strand breaks by polynucleotide ligase.

Transfection of Escherichia coli spheroplasts by native T5 phage DNA was not affected by treatment with polynucleotide ligase. Denatured T5 phage DNA infectivity, only 0.1% of the native DNA level, was increased slightly by polynucleotide ligase treatment. Renatured T5 phage DNA infectivity was also increased slightly by polynucleotide ligase treatment. To form an infective center with rec(+) spheroplasts, 1.6 to 2.1 native T5 phage DNA molecules were required; however, 1.4 T5 phage DNA molecules were required to form an infective center with recA(-)B(-) spheroplasts, and one molecule was sometimes sufficient for rec B(-) spheroplasts. Polynucleotide ligase treatment of T5 phage DNA had no effect on these parameters. Thus, the single-strand interruptions of T5 phage DNA are probably not essential to the survival of the parental T5 phage DNA, and T5 phage DNA, especially the denatured form, is highly sensitive to some nucleases in E. coli spheroplasts.

Coliphages↗