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Query: EC:6.5.1.2 (DNA ligase)
 2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Thermophilic and thermostable DNA ligase was purified to near homogeneity from the extract of Thermus thermophilus HB8. The purified enzyme has an isoelectric point at pH 6.6 and consists of a single polypeptide of about 79,000 in molecular weight on the bases of sodium dodecyl sulfate-polyacrylamide gel electrophoresis data and an equilibrium sedimentation method. The enzyme requires divalent cations, Mg2+ or Mn2+, and the optimum concentration of these ions being 5-9 X 10(-3) M and 3-6 X 10(-3) M, respectively. The enzyme also requires NAD as a cofactor. The apparent Km for NAD is 1.85 X 10(-8) M and that of (dT)10 is 1.4 X 10(-4) M. The pH optimum is 7.4-7.6 in Tris-HCl and 8.0 in collidine/HCl buffer. The joining reaction is activated by K+ and NH+4 at a concentration of 2-100 mM and inhibited by Na+ above 25 mM. The optimum temperatures of the joining of thymidylate oligomers in the presence of poly(dA) as a template are 27.5 degrees C for p(dT)s, 34.5 degrees C for p(dT)10, and 37 degrees C for p(dT)12-18 and that of cohesive-end DNA restriction fragments is 24-37 degrees C. The nick-closing activity of the enzyme was observed over a wide range of the temperature from 15 to 85 degrees C and the optimum temperature is 65-72 degrees C. The temperature dependency of ligation with HB8 DNA ligase for various substrates was found to shift to a region of 7-10 degrees C higher than that of T4 DNA ligase and the activity of HB8 DNA ligase decreased remarkably below 4 degrees C. The enzyme was stable for 1 week at 37 degrees C, its activity dropped by 50% within 2 days at 65 degrees C.
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PMID:Thermophilic DNA ligase. Purification and properties of the enzyme from Thermus thermophilus HB8. 646 54

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.
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PMID:Isolation of the messenger RNA for 8S DNA ligase in early developing axolotl egg and its cell free translation. 668 71

DNA ligase has been purified to near-homogeneity from the extract of bovine thymus with a yield of 5%. The purified enzyme catalyzed the joining of single-stranded breaks in duplex DNA at a rate of 33 nmol of phosphodiester bonds/min/mg of protein. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis and Ouchterlony double diffusion analysis. The enzyme is composed of a single polypeptide with a molecular weight of about 130,000. The enzyme has a Stokes radius of 52 A, a sedimentation coefficient of about 5 S, and a frictional ratio of 1.6. Apparent Km values for ATP and Mg2+ are 2 microM and 0.9 mM, respectively. Antibody against bovine thymus DNA ligase was prepared by injecting a rabbit with the purified enzyme. Immunochemical titrations revealed that the increased activity of DNA ligase observed after partial hepatectomy of rat and 16-fold higher activity level of mouse Ehrlich tumor cells compared with the host liver are due to a change in the enzyme quantity but not to a change in the catalytic efficiency of the enzyme molecule. Wide variations in the level of DNA ligase activity in extracts from various tissues of rat and mouse were accompanied by proportionate changes in the quantity of immunochemically reactive protein. The antibody inhibited DNA ligase activity from bovine tissues with 20-fold higher efficiency, compared with the enzyme from the rodent tissues. The enzyme activity from chick embryo was unaffected by the antibody.
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PMID:Eukaryotic DNA ligase. Purification and properties of the enzyme from bovine thymus, and immunochemical studies of the enzyme from animal tissues. 680 40

Three biochemically distinct DNA ligase activities have been identified in mammalian cell extracts. We have recently purified DNA ligase II and DNA ligase III to near homogeneity from bovine liver and testis tissue, respectively. Amino acid sequencing studies indicated that these enzymes are encoded by the same gene. In the present study, human and murine cDNA clones encoding DNA ligase III were isolated with probes based on the peptide sequences. The human DNA ligase III cDNA encodes a polypeptide of 862 amino acids, whose sequence is more closely related to those of the DNA ligases encoded by poxviruses than to replicative DNA ligases, such as human DNA ligase I. In vitro transcription and translation of the cDNA produced a catalytically active DNA ligase similar in size and substrate specificity to the purified bovine enzyme. The DNA ligase III gene was localized to human chromosome 17, which eliminated this gene as a candidate for the cancer-prone disease Bloom syndrome that is associated with DNA joining abnormalities. DNA ligase III is ubiquitously expressed at low levels, except in the testes, in which the steady-state levels of DNA ligase III mRNA are at least 10-fold higher than those detected in other tissues and cells. Since DNA ligase I mRNA is also present at high levels in the testes, we examined the expression of the DNA ligase genes during spermatogenesis. DNA ligase I mRNA expression correlated with the contribution of proliferating spermatogonia cells to the testes, in agreement with the previously defined role of this enzyme in DNA replication. In contrast, elevated levels of DNA ligase III mRNA were observed in primary spermatocytes undergoing recombination prior to the first meiotic division. Therefore, we suggest that DNA ligase III seals DNA strand breaks that arise during the process of meiotic recombination in germ cells and as a consequence of DNA damage in somatic cells.
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PMID:Mammalian DNA ligase III: molecular cloning, chromosomal localization, and expression in spermatocytes undergoing meiotic recombination. 756 92

A synthetic gene for a proteinase inhibitor (eglin C) that was obtained by direct amplification with oligonucleotides without using DNA ligase and polynucleotide kinase of T4 phage was cloned into expression vectors. A high yield of the polypeptide (110-130 mg/l) was attained in E. coli strains.
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PMID:[Chemical synthesis of a proteinase inhibitor (eglin C) gene without use of T4-DNA-ligase and its expression in E. coli]. 766 60

We have purified a high molecular weight complex (RC-1) from calf thymus nuclei that catalyzes a recombinational repair of double-strand gaps and deletions in DNA by gene conversion as well as cross-over events leading to cointegrant products. These have been detected by polymerase chain reaction analysis using oligonucleotide primer pairs that detect joined sequences originally present on only one or the other of the recombination substrates. RC-1 has an apparent molecular mass of about 550-600 kDa and contains at least five polypeptide chains: molecular masses about 230, 210, 160, 130, and 40 kDa. RC-1 contains a DNA polymerase, identified as DNA polymerase epsilon, that co-purifies with RC-1. A DNA ligase, most likely mammalian DNA ligase III, and a 5'-3' exonuclease also copurify with the RC-1. Most preparations of RC-1 contain low levels of a double-strand endonuclease, 3'-5' exonuclease and single-strand nuclease activities. However, DNA helicase, terminal deoxynucleotidyl transferase, or DNA topoisomerase I and II were not detected in RC-1. The DNA polymerase and DNA ligase in RC-1 can act in concert to repair a multiply gapped DNA to a covalently repaired duplex. The bovine single-strand-binding protein stimulates the formation of the recombination products and the repair reaction mentioned above about 4-fold.
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PMID:A mammalian protein complex that repairs double-strand breaks and deletions by recombination. 839 64

High-mobility-group 1 protein (HMG1) is an abundant eukaryotic DNA-binding protein, the cellular role of which remains ill-defined. To test the ability of HMG1 itself to mediate curvature in double-stranded DNA, we examined its effect on the phage T4 DNA ligase-dependent cyclization of short DNA fragments. HMG1 caused circle formation for fragments > or = 87 bp. Fragments of 123, 100, 92, and 87 bp did not cyclize in the absence of protein but formed covalently closed circular monomers efficiently in the presence of HMG1, indicating that the protein is capable of introducing bends into the duplex. The bending activity was maintained by a 79-amino acid polypeptide corresponding to a single HMG-box domain of HMG1. The binding affinity for the DNA minicircle was greater than for the corresponding linear fragment. These findings indicate that the role of HMG1 could involve both structure-specific recognition of prebent DNA and distortion of the DNA helix by bending and that the HMG-box domain may actually be responsible for this activity.
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PMID:High-mobility-group 1 protein mediates DNA bending as determined by ring closures. 841 24

The human DNA repair protein XRCC1 was overexpressed as a histidine-tagged polypeptide (denoted XRCC1-His) in Escherichia coli and purified in milligram quantities by affinity chromatography. XRCC1-His complemented the mutant Chinese hamster ovary cell line EM9 when constitutively expressed from a plasmid or when introduced by electroporation. XRCC1-His directly interacted with human DNA ligase III in vitro to form a complex that was resistant to 2 M NaCl. XRCC1-His interacted equally well with DNA ligase III from Bloom syndrome, HeLa and MRC5 cells, indicating that Bloom syndrome DNA ligase III is normal in this respect. Detection of DNA ligase III on far Western blots by radiolabelled XRCC1-His indicated that the level of the DNA ligase polypeptide was reduced approximately 4-fold in the mutant EM9 and also in EM-C11, a second member of the XRCC1 complementation group. Decreased levels of polypeptide thus account for most of the approximately 6-fold reduced DNA ligase III activity observed previously in EM9. Immunodetection of XRCC1 on Western blots revealed that the level of this polypeptide was also decreased in EM9 and EM-C11 (> 10-fold), indicating that the XRCC1-DNA ligase III complex is much reduced in the two CHO mutants.
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PMID:Characterization of the XRCC1-DNA ligase III complex in vitro and its absence from mutant hamster cells. 853 26

DNA ligase was highly purified from the fungus Coprinus cinereus at the miotic recombination stage, pachytene. The pachytene DNA ligase showed three polypeptides with molecular masses of 88, 84 and 80 kDa, as estimated by the [32P]AMP-labeling assay. These three polypeptides were susceptible to reaction with an mAb against a 16-amino-acid sequence in human DNA ligase I, which is conserved in C-terminal regions of mammalian, vaccinia virus and yeast DNA ligases. Since rapidly purified preparations from fresh pachytene cells exhibited a single polypeptide of DNA ligase with a molecular mass of 88 kDa, the smaller polypeptides seemed to be limited-degradation products of the 88-kDa polypeptide during the isolation and purification procedures. K(m) values for ATP and (dT)20 hybridized with (dA)n were 1.5 microM and 90 nM, respectively. This enzyme was capable of joining (dT)20.(rA)n and (rA)12-18 (dT)n as well as (dT)20.(dA)n and able to ligate blunt-ended DNA in the presence of poly(ethylene glycol) 6000. DNA ligases were also partially purified from zygotene cells at the meiotic pairing stage and mitotic mycelium cells. In their molecular mass, immuno-reactivity, K(m) value and substrate specificity, they were indistinguishable from pachytene DNA ligase. These results suggest that the fungus C. cinereus at the pachytene stage contains DNA ligase with a molecular mass of 88 kDa as a main or a single species, which is quite similar to DNA ligases from the zygotene and mycelium cells in molecular and catalytic properties.
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PMID:Characterization of DNA ligase from the fungus Coprinus cinereus. 864 14

Recombination protein complex RC-1, purified from calf thymus nuclear extracts, catalyzes cell-free DNA strand transfer and repair of gaps and deletions through DNA recombination. DNA polymerase E, DNA ligase III and a DNA structure-specific endonuclease co-purify with the five polypeptide complex. Here we describe the identification of two hitherto unknown subunits of RC-1. N-terminal amino acid sequences of the 160 and 130 kDa polypeptides display up to 100% identity to proteins of the structural maintenance of chromosomes (SMC) subfamilies 1 and 2. SMC proteins are involved in mitotic chromosome segregation and condensation, as well as in certain DNA repair pathways in fission (rad18 gene) and budding (RHC18 gene) yeast. The assignment was substantiated by immuno-cross-reactivity of the RC-1 subunits with polyclonal antibodies specific for Xenopus laevis SMC proteins. These antibodies, and polyclonal antibodies directed against the bovine 160 and 130 kDa polypeptides, named BSMC1 and BSMC2 (bovine SMC), inhibited RC-1-mediated DNA transfer, indicating that the SMC proteins are necessary components of the reaction. Two independent assays revealed DNA reannealing activity of RC-1, which resides in its BSMC subunits, thereby demonstrating a novel function of these proteins. To our knowledge, this is the first evidence for the association of mammalian SMC proteins with a multiprotein complex harboring, among others, DNA recombination, DNA ligase and DNA polymerase activities.
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PMID:SMC proteins constitute two subunits of the mammalian recombination complex RC-1. 867 Sep 10


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