Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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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)

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

Vaccinia virus DNA ligase has been expressed in Escherichia coli, purified, and biochemically characterized. The enzyme ligates double-stranded (ds) DNA substrates with either cohesive or blunt-end termini and the latter reaction is stimulated by PEG. Vaccinia virus DNA ligase can also ligate oligo(dT) when annealed to either a poly(dA) or a poly(rA) backbone and, remarkably, free oligo(dT). This ligation of a single-stranded (ss) substrate is unique among eukaryotic DNA ligases. The enzyme requires high ATP concentrations with a Km for the overall ligation of a ssDNA substrate of 0.8 mM. The salt, divalent cation, temperature, and pH requirements of the enzyme for the optimal ligation of ss and ds substrate are described.
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PMID:Ligation of double-stranded and single-stranded [oligo(dT)] DNA by vaccinia virus DNA ligase. 866 20

The purpose of this review is to summarize information published since 1990 on DNA replication, recombination and repair of vaccinia virus, a poxvirus. Temperature-sensitive mutations reveal four essential genes related to viral DNA replication: the E9L DNA polymerase, B1R protein kinase, D5R protein, and D4R uracil DNA glycosylase. Other proteins are likely to be also involved in viral DNA replication: the H6R DNA topoisomerase, I3L single stranded-DNA binding protein, H5R virosome-associated protein, and A50R DNA ligase. In addition, several viral-encoded proteins do regulate the level of the deoxyribonucleoside triphosphate pool: the J2R thymidine kinase, A48R thymidylate kinase, 14L and F4L subunits of ribonucleotide reductase, and F2L dUTPase. Despite the apparent simplicity of the mechanism of vaccinia virus DNA replication, several important questions related to the three Rs remain unsolved.
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PMID:Vaccinia virus DNA replication: a short review. 882 74

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.
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PMID:Domain structure of vaccinia DNA ligase. 901 21

The vaccinia virus D4R open reading frame, encoding the essential DNA repair enzyme uracil DNA glycosylase, was expressed in two permanent cell lines, the rabbit kidney cell line RK13 and the human fibroblast cell line 293. The temperature-sensitive vaccinia virus mutant ts4149, which maps within D4R, was able to grow under restrictive conditions in both of these transformed cell lines. Cell clones complemented D4R function to various degrees, demonstrating complementation of an essential vaccinia virus gene by a cell line constitutively expressing the essential function. Thus, the complementing host cells allowed the rescue of a virus defective in the D4R gene, demonstrating that this system may be used for the propagation of defective cytoplasmic DNA viruses. The defective virus grew to high yields only in the engineered cell lines. The data support the hypothesis that early gene products, such as uracil DNA glycosylase, supplied in trans can fully complement essential viral functions.
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PMID:Construction of a vaccinia virus deficient in the essential DNA repair enzyme uracil DNA glycosylase by a complementing cell line. 918 64

Vaccinia virus DNA ligase repairs nicked duplex DNA substrates consisting of a 5'-phosphate-terminated strand and a 3'-hydroxyl-terminated strand annealed to a bridging template strand. This study addresses the ability of vaccinia DNA ligase to seal nicked substrates containing one or more RNA strands. We found that the viral enzyme rapidly and efficiently joined a 3'-OH RNA to 5'-phosphate DNA when the reacting polynucleotides were annealed to a bridging DNA strand. In contrast, ligation of 3'-OH DNA to 5'-phosphate RNA was slow (0.2% of the rate of RNA-to-DNA ligation) and entailed the accumulation of high levels of RNA-adenylate intermediate. A native gel mobility shift assay showed that vaccinia DNA ligase discriminates at the substrate binding step between ligands containing 5'-phosphate DNA versus 5'-phosphate RNA at the nick. The enzyme displayed weak activity in RNA-to-RNA ligation on a bridging DNA template (0.01% of RNA-to-DNA activity). Vaccinia DNA ligase was incapable of joining two DNAs annealed on an RNA template. These results can be explained by a requirement for B-form helical conformation on the 5'-phosphate side of the nick. The robust RNA-to-DNA strand joining activity underscores the potential for vaccinia DNA ligase to catalyze RNA-based integration of host cell genetic information into the genome of cytoplasmic poxviruses.
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PMID:Ligation of RNA-containing duplexes by vaccinia DNA ligase. 922 Sep 97

Vaccinia virus DNA ligase has an intrinsic nick-sensing function. The enzyme discriminates at the substrate binding step between a DNA containing a 5' phosphate and a DNA containing a 5' hydroxyl at the nick. Further insights into nick recognition and catalysis emerge from studies of the active-site mutant K231A, which is unable to form the covalent ligase-adenylate intermediate and hence cannot activate a nicked DNA substrate via formation of the DNA-adenylate intermediate. Nonetheless, K231A does catalyze phosphodiester bond formation at a preadenylated nick. Hence, the active-site lysine of DNA ligase is not required for the strand closure step of the ligation reaction. The K231A mutant binds tightly to nicked DNA-adenylate but has low affinity for a standard DNA nick. The wild-type vaccinia virus ligase, which is predominantly ligase-adenylate, binds tightly to a DNA nick. This result suggests that occupancy of the AMP binding pocket of DNA ligase is essential for stable binding to DNA. Sequestration of an extrahelical nucleotide by DNA-bound ligase is reminiscent of the base-flipping mechanism of target-site recognition and catalysis used by other DNA modification and repair enzymes.
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PMID:Nick sensing by vaccinia virus DNA ligase requires a 5' phosphate at the nick and occupancy of the adenylate binding site on the enzyme. 937 33

Sequence analysis of the Lymantria dispar multicapsid nucleopolyhedrovirus (LdMNPV) genome identified an open reading frame (ORF) encoding a 548-amino-acid (62-kDa) protein that showed 35% amino acid sequence identity with vaccinia virus ATP-dependent DNA ligase. Ligase homologs have not been reported from other baculoviruses. The ligase ORF was cloned and expressed as an N-terminal histidine-tagged fusion protein. Incubation of the purified protein with [alpha-32P]ATP resulted in formation of a covalent enzyme-adenylate intermediate which ran as a 62-kDa labeled band on a sodium dodecyl sulfate-polyacrylamide gel. Loss of the radiolabeled band occurred upon incubation of the intermediate with pyrophosphate, poly(dA) . poly(dT)12-18, or poly(rA) . poly(dT)12-18, characteristics of a DNA ligase II or III. The protein was able to ligate a double-stranded synthetic DNA substrate containing a single nick and inefficiently ligated a 1-nucleotide (nt) gap but did not ligate a 2-nt gap. It was able to ligate short, complementary overhangs but not blunt-ended double-stranded DNA. In a transient DNA replication assay employing six plasmids containing the LdMNPV homologs of the essential baculovirus replication genes, a plasmid containing the DNA ligase gene was neither essential nor stimulatory. All of these results are consistent with the activity of type III DNA ligases, which have been implicated in DNA repair and recombination.
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PMID:Characterization of a baculovirus-encoded ATP-dependent DNA ligase. 976 60

The in vitro cloning of DNA molecules traditionally uses PCR amplification or site-specific restriction endonucleases to generate linear DNA inserts with defined termini and requires DNA ligase to covalently join those inserts to vectors with the corresponding ends. We have used the properties of Vaccinia DNA topoisomerase I to develop a ligase-free technology for the covalent joining of DNA fragments to suitable plasmid vectors. This system is much more efficient than cloning methods that require ligase because the rapid DNA rejoining activity of Vaccinia topoisomerase I allows ligation in only 5 min at room temperature, whereas the enzyme's high substrate specificity ensures a low rate of vector-alone transformants. We have used this topoisomerase I-mediated cloning technology to develop a process for accelerated cloning and expression of individual ORFs. Its suitability for genome-scale molecular cloning and expression is demonstrated in this report.
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PMID:Genome-scale cloning and expression of individual open reading frames using topoisomerase I-mediated ligation. 1020 60

Drug resistance is an obstacle preventing success of cancer chemotherapy. Resistance of vaccinia virus towards the topoisomerase II (topo II) targeting anti-cancer drug etoposide has been mapped to the viral DNA ligase gene. The present study was performed to elucidate if the DNA ligase activity, besides topo II levels, was altered in human lymphatic leukaemia cell strains with different levels of etoposide resistance. At measurements of DNA ligase activity with specific substrates, to distinguish between different DNA ligases, a reduced DNA ligase activity was observed in the resistant substrains. In contrast, the initial step of the ligation process, formation of DNA ligase--AMP complex, did not decrease in the resistant cell strains, suggesting an alteration in a later reaction leading to a deteriorated DNA ligation. The results suggest that decreased DNA ligase activity, besides topo II alterations, may contribute to etoposide resistance of the investigated CEM cells. The relevance of this finding will be further investigated.
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PMID:Reduced DNA ligase activity in etoposide resistant human lymphatic leukaemia CEM cells. 1184 1


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