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PubMed1985

DNA ligases as markers of lymphoid cell maturation and heterogeneity in the chicken.

The activities of 8 S and 6 S DNA ligases have been studied in the chicken lymphoid cells of blood, spleen and bursa of Fabricius at different stages of development, from late embryonic life to about 3 months after hatching. These cells have been sorted with the fluorescence-activated cell sorter FACS II on the basis of size and T or B antigenicity (immunofluorescence). The light 6 S DNA ligase has been previously demonstrated to be associated to a late stage of differentiation of thymocytes. In the bursa, a unique form of 8 S DNA ligase is found during the whole period of observation. This form of enzyme remains in the B cells of the spleen until 3 weeks after hatching, but is never present in the blood B cells. As far as T cells are concerned, the light DNA ligase is present in the blood from 18-day embryonic life on. In the spleen T cells, on the contrary, this enzyme appears only 3 weeks after hatching. Before this stage, splenic T cells are devoid of any form of DNA ligase activity. These findings show biochemical differences in T and B lymphocytes colonizing the periphery, blood and spleen, and suggest, at least for the T cells at early stages, a heterogeneity in the degree of differentiation.

Animals↗
PubMed1986

Periodic transcription as a means of regulating gene expression during the cell cycle: contrasting modes of expression of DNA ligase genes in budding and fission yeast.

Using cultures synchronised by three independent procedures, we have shown that the CDC9 gene, coding for DNA ligase, is periodically expressed in the Saccharomyces cerevisiae cell cycle. The level of CDC9 transcript increases many fold in late G1 reaching a peak at about the G1/S phase boundary and preceding the peak in histone message by some 20 min. The level of DNA ligase itself also fluctuates, showing the expected pattern for a stable enzyme synthesised periodically. In contrast, the transcript from the DNA ligase gene (CDC17) of Schizosaccharomyces pombe is present at a constant level throughout the cell cycle, and no fluctuation in amount was detected, although the histone H2A showed the expected periodic synthesis. Furthermore, DNA ligase activity remains at a constant level during the S. pombe cell cycle showing that there is unlikely to be any form of translational control. These contrasting modes of expression of the DNA ligase genes in the two organisms suggests that when periodic transcription is observed from an essential cell cycle gene, it may have no particular significance for regulating progress through the cell cycle. Also, regulatory circuits may be less well conserved between organisms than the processes they control and thus different organisms may utilise quite different modes of control to achieve the same ends.

Cell Cycle↗
PubMed1995

The N-terminal domain of human DNA ligase I contains the nuclear localization signal and directs the enzyme to sites of DNA replication.

DNA replication in mammalian cells occurs in discrete nuclear foci called 'replication factories'. Here we show that DNA ligase I, the main DNA ligase activity in proliferating cells, associates with the factories during S phase but displays a diffuse nucleoplasmic distribution in non-S phase nuclei. Immunolocalization analysis of both chloramphenicol acetyltransferase (CAT)-DNA ligase I fusion proteins and epitope tagged DNA ligase I mutants allowed the identification of a 13 amino acid functional nuclear localization signal (NLS) located in the N-terminal regulatory domain of the protein. Furthermore, the NLS is immediately preceded by a 115 amino acid region required for the association of the enzyme with the replication factories. We propose that in vivo the activity of DNA ligase I could be modulated through the control of its sub-nuclear compartmentalization.

3T3 Cells↗
PubMed1997

RNA splicing ligase activity in the archaeon Haloferax volcanii.

At least two separate enzymes, an endonuclease and a ligase, appear to be involved in tRNA splicing in halophilic archaea. We have identified and partially characterized a splicing ligase activity in cell extracts of Haloferax volcanii that can ligate deproteinized exon products generated in a separate endonuclease reaction. As in vitro transcribed partial intron-deleted derivative of H. volcanii elongator tRNA(Met) is used as substrate for the endonuclease. The ligase can also join the two exons that are independently eluted from the gels. This ligase activity is observed at a range (50 mM to 2.8 M) of monovalent cations in the assays, but is abolished when the enzyme preparations are depleted of the monovalent cations. In contrast, H. volcanii splicing endonuclease has been reported to require divalent cations and is inhibited by monovalent cations. Our endonuclease assays confirm these reports, and also show that the endonuclease is not permanently inactivated even in high monovalent cation containing extracts. The ligase activity in the extracts does not appear to require any divalent cation or exogenously added source of energy or phosphate.

Archaea↗
PubMed2002

Biochemical characterization of an ATP-dependent DNA ligase from the hyperthermophilic crenarchaeon Sulfolobus shibatae.

A gene encoding a putative ATP-dependent DNA ligase was identified in the genome of the hyperthermophilic archaeon Sulfolobus shibatae and expressed in Escherichia coli. The 601 amino acid recombinant polypeptide was a monomeric protein capable of strand joining on a singly nicked DNA substrate in the presence of ATP ( K(m)=34 micro mu) and a divalent cation (Mn(2+), Mg(2+), or Ca(2+)). dATP was partially active in supporting ligation catalyzed by the protein, but GTP, CTP, UTP, dGTP, dCTP, dTTP, and NAD(+) were inactive. The cloned Ssh ligase showed an unusual metal cofactor requirement; it was significantly more active in the presence of Mn(2+) than in the presence of Mg(2+) or Ca(2+). Unexpectedly, the native Ssh ligase preferred Mg(2+) and Ca(2+) rather than Mn(2+). Both native and recombinant enzymes displayed optimal nick-joining activity at 60-80 degrees C. Ssh ligase discriminated against substrates containing mismatches on the 3'-side of nick junction and was more tolerant of mismatches at the 5'-end than of those at the penultimate 5'-end. The enzyme showed little activity on a 1-nucleotide gapped substrate. This is the first biochemical study of a DNA ligase from the crenarchaeotal branch of the archaea domain.

Adenosine Triphosphate↗
PubMed2006

Characterization of an ATP-dependent DNA ligase from the acidophilic archaeon "Ferroplasma acidarmanus" Fer1.

Analysis of the genome of "Ferroplasma acidarmanus" Fer1, an archaeon that is an extreme acidophile, identified an open reading frame encoding a putative ATP-dependent DNA ligase, which we termed FaLig. The deduced amino acid sequence of FaLig contains 595 amino acids, with a predicted molecular mass of 67.8 kDa. "F. acidarmanus" Fer1 is classified as a Euryarchaeote, but phylogenetic analysis using amino acid sequences showed that FaLig is more similar to DNA ligases from Crenarchaeota, suggesting that lateral transfer of these genes has occurred among archaea. The gene sequence encoding FaLig was cloned into a bacterial expression vector harbouring an upstream His-tag to aid purification. Conditions for expression and purification from Escherichia coli were identified and recombinant FaLig was confirmed to be an ATP-dependent DNA ligase. Optimal conditions for nick-joining by the protein were pH 6-7, 0.5 mM ATP, in the presence of either Mg(2+) or Mn(2+). Using a range of nicked, double-stranded nucleic acids, ligation was detected with the same substrates as previously determined for other DNA ligases. Although FaLig is the DNA ligase from one of the most extreme acidophilic organism yet studied, this characterization suggests that its biochemical mechanism is analogous to that of enzymes from other cellular systems.

Adenosine Triphosphate↗
PubMed1981

Induction of DNA ligase during stimulation of DNA synthesis in intact rat liver by a dietary manipulation.

After a nutritional shift from a protein-free to a diet containing 50% casein, the activity of DNA ligase increases in intact rat liver in correlation with the induction of hepatic DNA replication. The treated rat liver as well as control rat liver contains a single species of DNA ligase having a sedimentation coefficient of about 5.5 S. The administration of cycloheximide in vivo completely inhibits the increase in DNA ligase activity and in DNA synthesis, indicating that DNA ligase is induced in the hepatic cells replicating DNA. In contrast to DNA ligase, DNA kinase is unchanged in the activity level by the dietary manipulation.

Animal Nutritional Physiological Phenomena↗
PubMed1987

Alterations in components of the ubiquitin-protein ligase system following maturation of reticulocytes to erythrocytes.

Previous studies have shown that the activity of the ubiquitin-mediated proteolytic system declines markedly following reticulocyte maturation, but the specific alterations responsible for this phenomenon have not been defined. We find that the rate of ATP-dependent degradation of 125I-albumin is reduced 20-fold in lysates of rabbit erythrocytes, as compared to reticulocyte lysates. The activity of the proteolytic system in erythrocyte extracts can be restored by supplementation with components of the ubiquitin-protein ligase system purified from reticulocytes by affinity chromatography. These components are the ubiquitin-carrier protein E2, the activity of which is nearly completely absent, and the ligase E3, the activity of which is partially reduced in erythrocytes. Erythrocyte extracts contain other ligases which attach a single, or a few ubiquitin molecules to proteins; these products are different from the multi-ubiquitin derivatives which are formed by the ligase system of protein breakdown. Mature red cells may thus serve to distinguish between different ubiquitin-protein ligase systems with presumably different functions.

Adenosine Triphosphate↗
PubMed1994

Xenobiotic acyl-CoA formation: evidence of kinetically distinct hepatic microsomal long-chain fatty acid and nafenopin-CoA ligases.

Multiplicity of hepatic microsomal coenzyme A ligases catalyzing acyl-CoA thioester formation is an important factor for consideration in relation to the metabolism of xenobiotic carboxylic acids. In this study the kinetic characteristics of rat hepatic microsomal nafenopin-CoA ligase were studied and compared with those of long-chain fatty acid (palmitoyl) CoA ligase. The high affinity component of palmitoyl-CoA formation was inhibited by nafenopin (Ki 53 microM) and ciprofibrate (Ki 1000 microM). Analagous to palmitoyl-CoA, nafenopin-CoA formation was catalyzed by an apparent high affinity low capacity isoform (Km 6 +/- 2.5 microM, Vmax 0.33 +/- 0.12 nmol/mg per min) which was inhibited competitively by palmitic acid (mean Ki 1.7 microM, n = 5) and R-ibuprofen (mean Ki 10.8 microM, n = 5) whilst ciprofibrate and clofibric acid were ineffective as inhibitors. The intrinsic metabolic clearance of nafenopin to nafenopin-CoA (Vmax/Km 0.057 +/- 0.011 nmol/mg/min/ +/- M) was similar to that reported recently for the formation of ibuprofenyl-CoA by rat liver microsomes. Evidence of both a substantial difference between the Km and Ki for nafenopin and lack of commonality with regard to xenobiotic inhibitors suggests that the high affinity microsomal nafenopin-CoA and long-chain fatty acid-CoA ligases are kinetically distinct. Thus until the current 'long-chain like' xenobiotic-CoA ligases are fully characterised in terms of substrate specificity, inhibitor profile, etc, it will be impossible to rationalize (and possibly predict) the metabolism and hence toxicity of xenobiotic carboxylic acids forming acyl-CoA thioester intermediates.

Acyl Coenzyme A↗
PubMed1983

DNA ligase in Axolotl egg: a model for study of gene activity control.

Replacement of the light form of DNA ligase (6 S) by the heavy form (8 S) in activated egg of Axolotl has been studied as a model for change in genetic activity exerted by the female pronucleus. Nuclear transplantation shows that a blastula nucleus is able to govern the replacement of the light ligase by the heavy one. The result is not the same if the grafted nucleus is taken from an androgenetic embryo, devoid of the heavy enzyme. Therefore the change in the properties of the female pronucleus appears stable and autoreproducible. Gamma irradiations delivered at different times after activation establish that the replacement of the ligase forms depends on an intact nucleus up to 3 hr 30 min after activation, and thereafter is achieved independently of any nuclear damage. Inhibitors of DNA replication impede the change of enzymatic form in reversible process, suggesting new chromatin synthesis as prerequisite for expression of the new genetic activity. The quantitative level of DNA ligase activity does not show any dose effect when one or many nuclei are present in the same cytoplasm. However, a change in nucleotide concentration results in a change in DNA ligase activity, indicating cytoplasmic control of enzymatic regulation.

Ambystoma↗
PubMed1990

Blunt-end and single-strand ligations by Escherichia coli ligase: influence on an in vitro amplification scheme.

A ligase-based, in vitro DNA amplification system (LAR) has been described by Wu and Wallace [Genomics 4 (1989) 560-569]. This strategy is based on the ability of a DNA ligase to join the 5' phosphate of one DNA molecule to the 3' hydroxyl of a second during a nick-closing reaction. Escherichia coli DNA ligase has been used in place of the T4 DNA ligase in our study in order to limit template-independent ligation activities, which lower the sensitivity of this amplification procedure. The results of this study indicate that E. coli ligase also joins blunt-ended DNA molecules and some single-stranded oligodeoxyribonucleotides, in the absence of a complementary template, with an efficiency which is sensitive to both the concentrations of DNA substrate and enzyme.

Base Sequence↗
PubMed2004

Distinct pools of proliferating cell nuclear antigen associated to DNA replication sites interact with the p125 subunit of DNA polymerase delta or DNA ligase I.

Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication, repair, and cell cycle control. PCNA is a homotrimeric ring that, when encircling DNA, is not easily extractable. Consequently, the dynamics of protein-protein interactions established by PCNA at DNA replication sites is not well understood. We have used DNase I to release DNA-bound PCNA together with replication proteins including the p125-catalytic subunit of DNA polymerase delta (p125-pol delta), DNA ligase I, cyclin A, and cyclin-dependent kinase 2 (CDK2). Interaction with these proteins was investigated by immunoprecipitation with antibodies binding near the interdomain connector loop or to the C-terminal domain of PCNA, respectively, or with antibodies to p125-pol delta or DNA ligase I. PCNA interaction with p125-pol delta or DNA ligase I was detected only by the latter antibodies, and found to be mutually exclusive. In contrast, antibodies to PCNA co-immunoprecipitated only CDK2. A GST-p21(waf1/cip1) C-terminal peptide displaced p125-pol delta and DNA ligase I, but not CDK2, from PCNA. These results suggest that PCNA trimers bound to DNA during the S phase are organized as distinct pools able to bind selectively different partners. Among them, p125-pol delta and DNA ligase I interact with PCNA in a mutually exclusive manner.

Antibodies↗
PubMed2006

Human DNA ligases I, III, and IV-purification and new specific assays for these enzymes.

The joining of DNA strand breaks by DNA ligases is required to seal Okazaki fragments during DNA replication and to complete almost all DNA repair pathways. In human cells, there are multiple species of DNA ligase encoded by the LIG1, LIG3, and LIG4 genes. Here we describe protocols to overexpress and purify recombinant DNA ligase I, DNA ligase IIIbeta, and DNA ligase IV/XRCC4 and the assays used to purify and distinguish between these enzymes. In addition, we describe a fluorescence-based ligation assay that can be used for high throughput screening of chemical libraries.

Base Sequence↗
PubMedDate not supplied

DNA ligase activity in carcinogen-treated human fibroblasts.

In an enzymological approach to study DNA repair mechanisms induced by carcinogen-treatment of mammalian cells, we have investigated how DNA ligase activity is affected by the treatment with several compounds producing different DNA lesions. Stationary cultures of human fibroblasts were exposed to various doses of carcinogens (UV-light at 254 nm, N-acetoxy-acetyl-aminofluorene, ethyl-methane sulfonate, N-methylnitro-nitrosoguanidine, mitomycin C and 4-nitroquinoline-N-oxide) at different time-intervals before preparing crude cellular extracts and assaying for ligase activity. Results have shown that: 1. UV-irradiation, AAAF, 4NQO or MMC treatment of cells induces a two-fold increase in the ligase activity compared to control cells within 48 hours following the treatment. 2. A partial purification of the enzyme from these cellular crude extracts by sedimentation through sucrose gradients has shown: a. DNA ligase activity from control cells presents a profile composed of two distinct peaks sedimenting respectively at about 4S and 7S; b. the carcinogen treatment of either repair-proficient human fibroblasts or repair-deficient xeroderma pigmentosum cells (complementation group A) seems to induce a specific increase of the 4S-form of DNA ligase.

Carcinogens↗
PubMed2000

Lif1p targets the DNA ligase Lig4p to sites of DNA double-strand breaks.

DNA ligases catalyse the joining of DNA single- and double-strand breaks. Saccharomyces cerevisiae Cdc9p is a homologue of mammalian DNA ligase I and is required for DNA replication, recombination and single-strand break repair. The other yeast ligase, Lig4p/Dnl4p, is a homologue of mammalian DNA ligase IV, and functions in the non-homologous end-joining (NHEJ) pathway of DNA double-strand break repair [1] [2] [3] [4]. Lig4p interacts with Lif1p, the yeast homologue of the human ligase IV-associated protein, XRCC4 [5]. This interaction takes place through the carboxy-terminal domain of Lig4p and is required for Lig4p stability. We show that the carboxy-terminal interaction region of Lig4p is necessary for NHEJ but, when fused to Cdc9p, is insufficient to confer NHEJ function to Cdc9p. Also, Lif1p stimulates the in vitro catalytic activity of Lig4p in adenylation and DNA ligation. Nevertheless, Lig4p is inactive in NHEJ in the absence of Lif1p in vivo, even when Lig4p is stably expressed. We show that Lif1p binds DNA in vitro and, through in vivo cross-linking and chromatin immuno precipitation assays, demonstrate that it targets Lig4p to chromosomal DNA double-strand breaks. Furthermore, this targeting requires another key NHEJ protein, Ku.

Antigens, Nuclear↗
PubMed1998

DNA ligase IV is essential for V(D)J recombination and DNA double-strand break repair in human precursor lymphocytes.

Nonhomologous DNA end joining (NHEJ) is the major pathway for repairing double-strand DNA breaks. V(D)J recombination is a double-strand DNA breakage and rejoining process that relies on NHEJ for the joining steps. Here we show that the targeted disruption of both DNA ligase IV alleles in a human pre-B cell line renders the cells sensitive to ionizing radiation and ablates V(D)J recombination. This phenotype can only be reversed by complementation with DNA ligase IV but not by expression of either of the remaining two ligases, DNA ligase I or III. Hence, DNA ligase IV is the activity responsible for the ligation step in NHEJ and in V(D)J recombination.

Alleles↗
PubMed1982

Reversal of T4 RNA ligase.

Unexpected products detected in oligoribonucleotide synthesis reactions catalyzed by T4 RNA ligase are shown to be a result of a partial reversal of the enzyme reaction. A transfer assay for the reversal of the third step in the RNA ligase reaction mechanism and an exchange assay for the reversal of both the second and third steps are described. Reversal is confirmed by the formation of the expected covalent intermediates, adenylylated donor and adenylyl ligase, from a reaction containing 5'-AMP, unadenylylated ligase, and the tetranucleotide (Ap)3Cp. In the reverse reaction, RNA ligase shows a strong preference for hydrolysis of the 3'-terminal phosphodiester bonds of oligoribonucleotides which terminate in a 3'-phosphate. Several strategies are discussed to minimize the effects of reversal in the enzymatic synthesis of oligoribonucleotides.

Adenosine Monophosphate↗
PubMed1980

T4 ribonucleic acid ligase joins single-strand oligo(deoxyribonucleotides).

T4 RNA ligase joins a 3'-hydroxyl-terminated acceptor oligoribonucleotide to a 5'-phosphate-terminated donor oligoribonucleotide. An analogous reaction with single-strand DNA oligonucleotides would be useful for the synthesis of defined sequences of DNA because it would eliminate the need to synthesize complementary sequences to form the duplex substrates required by DNA ligase. We have studied the model reaction dA(pdA)5 + [5'-32P] (pdT)4pdCp leads to dA(pdA)5 [3' leads to 5'-32P]pdT(pdT)3pdCp and have obtained 40-60% yields at equimolar concentrations (100 microM to 1 mM) of the two substrates. Higher yields have been obtained when acceptor concentrations in excess of those of the donor are used. The use of a 5'-hydroxyl, 3'-hydroxyl terminated acceptor and a 5'-phosphate, 3'-phosphate terminated donor limits the reaction to a unique product. The 3'-phosphate-terminated donor was prepared by using RNA ligase to add a single deoxyribonucleoside 3',5'-bisphosphate donor to an oligo(deoxyribonucleotide) acceptor [Hinton, D.M., Baez, J.A., & Gumport, R.I. (1978) Biochemistry 17, 5091]. The DNA oligomer joining reaction requires low concentrations of ATP and an ATP regenerating system, Mn2+, high levels of nuclease-free RNA ligase (30 microM), and incubation for several days at 17 degrees C. The product of the reaction was characterized by its resistance to alkaline phosphatase, degradation by micrococcal nuclease to the expected product [3'-32P]dAMP, and mobility during high-pressure liquid chromatography on RPC-5. The joining of several other deoxyoligomers was also demonstrated. We anticipate that this reaction of RNA ligase will contribute to its usefulness as a reagent for the synthesis of DNA of defined sequence.

DNA, Single-Stranded↗