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PubMed1980

T4 RNA ligase as a nucleic acid synthesis and modification reagent.

Oligodeoxyribonucleotides corresponding to portions of the recognition sequence and analogues thereof of the Eco RI restriction endonuclease have been synthesized using T4 RNA ligase. The successive addition of deoxyribonucleoside-3',5'-bisphosphates to preformed deoxyoligomers allowed stepwise oligodeoxyribonucleotide synthesis. Single strand deoxyoligomers were also joined to one another by the enzyme. In addition, biotin, and fluorophore tetramethylrhodamine, and hexylamine have been added to RNA via an ATP-independent RNA ligase reaction using their ADP adducts as substrates. When the beta-substituent on ADP is a good leaving group, e.g. p-nitrophenol or 4-methylumbelliferol, the RNA product is the 2'-(3')-cyclicphosphate derivative.

DNA Restriction Enzymes↗
PubMedDate not supplied

[Isolation of highly purified RNA ligase from bacteriophage T4].

A technique for isolation of RNA-ligase of bacteriophage T4 was proposed. It is mainly based on the using of Soviet materials and sorbents and includes seven purification stages. The technique enables to isolate about 80 000 units of active enzyme from 100 g of E. coli B cells infected with the phage. T4am N82; that makes up 20% of the activity of the cell extract. The obtained preparations of RNA-ligase are homogeneous by the data of electrophoresis and practically, free of endo- and exonuclease admixtures.

Chromatography, Affinity↗
PubMedDate not supplied

[Purification and characteristics of an RNA-ligase preparation from bacteriophage T4].

A purification techniques was developed to obtain a preparation of the bacteriophage T4 RNA-ligase (EC 6.5.1.3) free of contaminating nuclease activities. It includes fractioning on phosphocellulose and DEAE-cellulose, chromatography on DEAE-cellulose, isoelectric sedimentation, gel filtration through Sephadex G-100 and chromatography on hydroxylapatite and aminohexyl-sepharose. The enzyme yield amounts to 30-37%. By means of biochemical tests, the preparation of RNA-ligase was found to be good for molecular biology and gene engineering, in particular for such areas where oligodeoxyribonucleotides and high molecular-weight RNA are used as substrates. The ligating ability of the enzyme was demonstrated by means of the preparative synthesis of nonaribonucleotide ApUpG(pU)6 from trinucleoside diphosphate ApUpG and hexoribouridylic acid, as well as by insertion of [5'-32P]-labelled cytidine-3',5'-diphosphate in the 3'-end of the bacteriophage MS 2 RNA.

Chromatography, DEAE-Cellulose↗
PubMed1996

A Candida albicans gene encoding a DNA ligase.

A DNA ligase-encoding gene (Ca CDC9) was cloned from Candida albicans by complementation of an ime-1 mutation in Saccharomyces cerevisiae. In this system, IME1 function was assayed using a S. cerevisiae strain with a ime2-promoter-lacZ gene fusion such that following transformation with a C. albicans genomic library, the presence of positive clones was indicated upon the addition of X-gal to sporulation media. Transforming fragments were subcloned in pGEM7 and sequenced. Sequence homology with several ATP-dependent DNA ligases from viruses, fission yeast, human, baker yeast and bacteria was observed.

Amino Acid Sequence↗
PubMed1994

Enhanced repair of a cisplatin-damaged reporter chloramphenicol-O-acetyltransferase gene and altered activities of DNA polymerases alpha and beta, and DNA ligase in cells of a human malignant glioma following in vivo cisplatin therapy.

Current evidence suggest an important role for increased repair of drug-induced DNA damage as one of the major mechanisms involved in tumor cell resistance to cis-DDP. In this study, we examined the DNA repair capacity and the activities of three DNA repair related proteins, namely, DNA polymerases alpha and beta, and total DNA ligase in cells of a malignant oligodendroglioma obtained from a patient before therapy and compared it with those of a specimen of the tumor acquired after the patient had failed cis-DDP therapy. DNA repair capacity was quantitated as the extent of reactivation of the chloramphenicol-O-acetyltransferase (CAT) gene in a eukaryotic expression vector that had been damaged and inactivated by prior treatment with cis-DDP and then transfected into the tumor cells. The extent of DNA-platinum adduct formation in the expression vector was determined by flameless atomic absorption spectrometry. The level of cis-DDP resistance of cells of the two tumors was determined with the capillary tumor stem cell assay. We observed a 2.8-fold increased capacity to repair Pt-DNA adducts and reactivate the CAT gene in cells of the tumor obtained after cis-DDP therapy, compared to cells of the untreated tumor. This was associated with increases of 9.4-fold and a 2.3-fold, respectively, in DNA polymerase beta and total DNA ligase activities in cells of the treated tumor. At 5 microM cis-DDP, there was a 5.9-fold increase in the in vitro cis-DDP resistance of post-therapy tumor cells relative to cells of the untreated tumor.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Line↗
PubMed1996

Analysis of ligase chain reaction products via matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry.

A rapid and accurate detection of ligation products generated in ligase chain reactions (LCR) by using matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI-TOF-MS) is reported. LCR with Pfu DNA ligase was performed with a wild-type template and a template carrying a single point mutation within the Escherichia coli lacI gene as a model system. Starting from about 1 fmol of template DNA the ligation product generated in the positive reactions was analyzed with HPLC and MALDI-TOF-MS, whereby the need of proper sample purification prior to mass spectrometric analysis was demonstrated. A purification procedure with a high potential for automation using streptavidin-coated magnetic particles and ultrafiltration was introduced. Plasmid DNA and short single-stranded oligonucleotides have been used as template. A point mutation could be discriminated from the wild-type template due to the absence or presence of ligation product. This approach allows the rapid-specific detection of template DNA in femtomole amounts and moreover can distinguish between sequence variations in DNA molecules down to point mutations without the need for labeling, gel electrophoresis, membrane transfer, or hybridization procedures.

Base Sequence↗
PubMed1999

Functional domains of an NAD+-dependent DNA ligase.

Limited proteolysis of the NAD+-dependent DNA ligase from Bacillus stearothermophilus with thermolysin results in two fragments which were resistant to further proteolysis. These fragments were characterised by N-terminal protein sequencing and electrospray mass spectrometry. The larger, N-terminal fragment consists of the first 318 residues and the smaller, C-terminal fragment begins at residue 397 and runs to the C terminus. Both fragments were over-expressed in Escherichia coli and purified to homogeneity from this source. The large fragment retains the full self-adenylation activity of the intact enzyme, has minimal DNA binding activity and vastly reduced ligation activity. The small fragment lacks adenylation activity but binds to nicked DNA with a similar affinity to that of the intact enzyme. It is unable to stimulate the ligation activity of the large fragment. Atomic absorption spectroscopy showed that the intact protein and the small fragment bind a zinc ion but the large fragment does not. No evidence of any interaction between the two fragments could be obtained. Thus, we conclude that NAD+-dependent DNA ligases consist of at least two discrete functional domains: an N-terminal domain which is responsible for cofactor binding and self adenylation, and a C-terminal DNA-binding domain which contains a zinc binding site.

Adenine↗
PubMed1993

Purification and characterization of phenylacetate-coenzyme A ligase from a denitrifying Pseudomonas sp., an enzyme involved in the anaerobic degradation of phenylacetate.

The enzyme catalysing the first step in the anaerobic degradation pathway of phenylacetate was purified from a denitrifying Pseudomonas strain KB 740. It catalyses the reaction phenylacetate + CoA + ATP-->phenylacetyl-CoA + AMP + PPi and requires Mg2+. Phenylacetate-CoA ligase (AMP forming) was found in cells grown anaerobically with phenylacetate and nitrate. Maximal specific enzyme activity was 0.048 mumol min-1 x mg-1 protein in the mid-exponential growth phase. After 640-fold purification with 18% yield, a specific activity of 24.4 mumol min-1 mg-1 protein was achieved. The enzyme is a single polypeptide with Mr of 52 +/- 2 kDa. The purified enzyme shows high specificity towards the aromatic inducer substrate phenylacetate and uses ATP preferentially; Mn2+ can substitute for Mg2+. The apparent Km values for phenylacetate, CoA, and ATP are 60, 150, and 290 microM, respectively. The soluble enzyme has an optimum pH of 8.5, is insensitive to oxygen, but is rather labile and requires the presence of glycerol and/or phenylacetate for stabilization. The N-terminal amino acid sequence showed no homology to other reported CoA-ligases. The expression of the enzyme was studied by immunodetection. It is present in cells grown anaerobically with phenylacetate, but not with mandelate, phenylglyoxylate, benzoate; small amounts were detected in cells grown aerobically with phenylacetate.

Adenosine Monophosphate↗
PubMed1990

The VANA glycopeptide resistance protein is related to D-alanyl-D-alanine ligase cell wall biosynthesis enzymes.

Inducible resistance to the glycopeptide antibiotics vancomycin and teicoplanin is mediated by plasmid pIP816 in Enterococcus faecium strain BM4147. Vancomycin induced the synthesis of a ca. 40 kDa membrane-associated protein designated VANA. The resistance protein was partially purified and its N-terminal sequence was determined. A 1761 bp DNA restriction fragment of pIP816 was cloned into Escherichia coli and sequenced. When expressed in E. coli, this fragment encoded a ca. 40 kDa protein that comigrated with VANA from enterococcal membrane fractions. The ATG translation initiation codon for VANA specified the methionine present at the N-terminus of the protein indicating the absence of signal peptide processing. The amino acid sequence deduced from the sequence of the vanA gene consisted of 343 amino acids giving a protein with a calculated Mr of 37,400. VANA was structurally related to the D-alanyl-D-alanine (D-ala-D-ala) ligases of Salmonella typhimurium (36% amino acid identity) and of E. coli (28%). The vanA gene was able to transcomplement an E. coli mutant with thermosensitive D-ala-D-ala ligase activity. Thus, the inducible resistance protein VANA was structurally and functionally related to cytoplasmic enzymes that synthesize the target of glycopeptide antibiotics. Based on these observations we discuss the possibility that resistance is due to modification of the glycopeptide target.

Amino Acid Sequence↗
PubMed1992

Temperature sensitivity of the cdc9-1 allele of Saccharomyces cerevisiae DNA ligase is dependent on specific combinations of amino acids in the primary structure of the expressed protein.

In this study we present the characterization of the temperature-sensitive mutant allele cdc9-1 encoding DNA ligase, of Saccharomyces cerevisiae strain A364A by DNA sequencing. Comparison with the published wild-type sequence from strain SK1 revealed 13 nucleotide exchanges between these two sequences, which are derived from non-isogenic genetic backgrounds. Only four of these changes, distributed over the whole coding region, lead to amino acid exchanges in the protein chain. Our analysis of the sequence of the wild-type CDC9 allele from strain A364A revealed differences from the isogenic cdc9-1 allele in only two nucleotides: one silent change and one leading to a single amino acid exchange. The latter is therefore responsible for the temperature-sensitive phenotype. A mosaic protein, in which a region carrying this amino acid exchange has been inserted in place of the corresponding part of CDC9 from the non-isogenic strain SK1, is not temperature sensitive. The exchange of a longer stretch of DNA leading to atteration of three amino acids of the protein compared with the original sequence of SK1 is required to obtain a temperature-sensitive DNA ligase in this strain, while in strain A364A a single amino acid change is sufficient for expression of a temperature-sensitive protein.

Amino Acid Sequence↗
PubMed2001

Reduced detection of Chlamydia trachomatis by the ligase chain reaction assay due to suboptimal storage of urine.

Detection of Chlamydia trachomatis by the ligase chain reaction assay was assessed in urine samples that had been stored at 4 degrees C and at ambient temperature for 6-10 days before testing. Six of 67 (9%) ligase chain reaction-positive urine samples stored at 4 degrees C and 5 of 29 (17%) stored at ambient temperature became negative, a difference that is not statistically significant. Most of the urine samples that were negative after storage contained a small number of chlamydial elementary bodies, and almost three-quarters of them were from women. Optimal pretest storage conditions for urine samples should be maintained if the maximum benefit is to be obtained from this highly sensitive assay.

Chlamydia Infections↗
PubMed1976

Joining of simian virus 40 DNA molecules at endonuclease R Eco Ri sites by polynucleotide ligase and analysis of the products by agarose gel electrophoresis.

DNA molecules cut with endonuclease R Eco Ri can be joined at Eco Ri cleavage sites by incubation with polynucleotide ligase. In order to define the optimum conditions for this reaction, linear Simian Virus 40 DNA molecules (SV40(Lri)) produced by endonuclease R Eco Ri cleavage of SV40 form i DNA were joined using polynucleotide ligases specified by bacteriophage T4 and Escherichia coli. We have determined that the concentration of the substrate DNA molecules is the most important factor determining the distribution of covalently joined product molecules into a variety of circular and linear monomeric and oligomeric species.

DNA Restriction Enzymes↗
PubMed1981

Chromatin conformation modulates repair of single strand interruptions by polynucleotide ligase-[3H] AMP.

Conformationally distinct chromatin populations were utilized as substrates to quantitate the relative amount of and accessibility of internal 5'-phosphomonoester breaks in DNA-chromatin. In these studies, a constant amount of chromatin as well as deproteinized DNA derived from the respective chromatin sample was titrated with increasing quantities of adenylated polynucleotide ligase intermediate. This enzyme intermediate releases its AMP moiety while repairing a DNA single strand interruption, release of AMP being directly proportional to the number of internal 5'-phosphomonoester breaks repaired. Results of this study indicate that the ability of polynucleotide ligase to repair DNA breaks within chromatin was affected by the conformational state of the chromatin. The degree of conformational constraint present in a given chromatin, therefore, determined the capacity of the enzyme to repair internal DNA 5'-phosphomonoester breaks.

Adenosine Monophosphate↗
PubMed1995

Isolation of a ribozyme with 5'-5' ligase activity.

BACKGROUND: Many new ribozymes, including sequence-specific nucleases, ligases and kinases, have been isolated by in vitro selection from large pools of random-sequence RNAs. We are attempting to use in vitro selection to isolate new ribozymes that have, or can be evolved to have, RNA polymerase-like activities. As phosphorimidazolide-activated nucleosides are extensively used to study non-enzymatic RNA replication, we wished to select for a ribozyme that would accelerate the template-directed ligation of 5'-phosphorimidazolide-activated oligonucleotides. RESULTS: Ribozymes selected to perform the desired template-directed ligation reaction instead ligated themselves to the activated substrate oligonucleotide via their 5'-triphosphate, generating a 5'-5' P1,P4-tetraphosphate linkage. Deletion analysis of one of the selected sequences revealed that a 54-nucleotide RNA retained activity; this small ribozyme folds into a pseudoknot secondary structure with an internal binding site for the substrate oligonucleotide. The ribozyme can also synthesize 5'-5' triphosphate and 5'-5' pyrophosphate linkages. CONCLUSIONS: The emergence of ribozymes that accelerate an unexpected 5'-5' ligation reaction from a selection designed to yield template-dependent 3'-5' ligases suggests that it may be much easier for RNA to catalyze the synthesis of 5'-5' linkages than 3'-5' linkages. 5'-5' linkages are found in a variety of contexts in present-day biology. The ribozyme-catalyzed synthesis of such linkages raises the possibility that these 5'-5' linkages originated in the biochemistry of the RNA world.

Base Sequence↗
PubMed2006

DNA point mutation detection based on DNA ligase reaction and nano-Au amplification: a piezoelectric approach.

A novel piezoelectric method for DNA point mutation detection based on DNA ligase reaction and nano-Au-amplified DNA probes is proposed. A capture probe was designed with the potential point mutation site located at the 3' end and a thiol group at the 5' end to be immobilized on the gold electrode surface of quartz crystal microbalance (QCM). Successive hybridization with the target DNA and detection probe of nano-Au-labeled DNA forms a double-strand DNA (dsDNA). After the DNA ligase reaction and denaturing at an elevated temperature, the QCM frequency would revert to the original value for the target with single-base mismatch, whereas a reduced frequency response would be obtained for the case of the perfect match target. In this way, the purpose of point mutation discrimination could be achieved. The current approach is demonstrated with the identification of a single-base mutation in artificial codon CD17 of the beta-thalassemia gene, and the wild type and mutant type were discriminated successfully. The scanning electron microscope (SEM) image showing that plenty of gold nanoparticles remained on the electrode surface demonstrated that the nano-Au label served as an efficient signal amplification agent in QCM assay. A detection limit of 2.6 x 10(-9)mol/L of oligonucleotides was achieved. Owing to its ease of operation and low detection limit, it is expected that the proposed procedure may hold great promise in both research-based and clinical genomic assays.

Base Pair Mismatch↗
PubMed2003

The RING-H2 protein RNF11 is differentially expressed in breast tumours and interacts with HECT-type E3 ligases.

A breast cancer-associated mRNA originally cloned as a 475-bp partial cDNA from a library enriched for tumour cDNAs [Oncogene 16 (1998) 327] is expressed at high levels in breast and prostate cancer cells. Immunohistochemical analysis indicates that the protein is expressed in primary breast tumours. We used RT-PCR to generate a full-length 2852 nt mRNA sequence that includes the hypothetical open reading frame (ORF) for human RNF11. Our analysis shows that RNF11 encodes modular domains and motifs likely to interact with other proteins involved in oncogenesis. Chief among these are the RING-H2 finger domain that could facilitate the degradation of specific substrate(s) involved in oncogenesis and the PY motif which binds to WW-domain proteins, several of which are known to be E3 ubiquitin ligases. Our GST-pulldown and immunoprecipitation results indicate that RNF11 interacts with the E3 ligase AIP4 when coexpressed with RNF11 in mammalian cells.

Amino Acid Sequence↗
PubMed2005

Analysis of ligation and DNA binding by Escherichia coli DNA ligase (LigA).

NAD(+)-dependent DNA ligases are essential enzymes in bacteria, with the most widely studied of this class of enzymes being LigA from Escherichia coli. NAD(+)-dependent DNA ligases comprise several discrete structural domains, including a BRCT domain at the C-terminus that is highly-conserved in this group of proteins. The over-expression and purification of various fragments of E. coli LigA allowed the investigation of the different domains in DNA-binding and ligation by this enzyme. Compared to the full-length protein, the deletion of the BRCT domain from LigA reduced in vitro ligation activity by 3-fold and also reduced DNA binding. Using an E. coli strain harbouring a temperature-sensitive mutation of ligA, the over-expression of protein with its BRCT domain deleted enabled growth at the non-permissive temperature. In gel-mobility shift experiments, the isolated BRCT domain bound DNA in a stable manner and to a wider range of DNA molecules compared to full LigA. Thus, the BRCT domain of E. coli LigA can bind DNA, but it is not essential for DNA nick-joining activity in vitro or in vivo.

Base Sequence↗
PubMed2005

The U-box ligase carboxyl-terminus of Hsc 70-interacting protein ubiquitylates Epsin.

Epsin is an endocytic adaptor protein involved in the regulation of clathrin-dependent endocytosis. We and others have demonstrated that Epsin is ubiquitylated in cells and requires its ubiquitin interacting motifs (UIMs) for this modification. To further elucidate the mechanism of Epsin ubiquitylation, we initiated studies to identify the E3 ligase(s) that modifies Epsin. In this study, we discovered that the U-box ubiquitin ligase carboxyl-terminus of Hsc70 interacting protein (CHIP) ubiquitylated Epsin. Using an in vitro ubiquitylation assay, we demonstrate that CHIP specifically ubiquitylated Epsin in a UIM-dependent manner. Furthermore, overexpression of CHIP in cells increased Epsin ubiquitylation also in a UIM-dependent manner. Together, these data provide evidence that CHIP functions to ubiquitylate the endocytic protein Epsin.

Adaptor Proteins, Vesicular Transport↗