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)

We have investigated the effect on the DNA structure of protein MC1, a basic and small polypeptide (Mr 10700) representing the major chromosomal protein in Methanosarcinaceae. The ability of protein MC1 to strongly favour cyclization upon polymerization of short DNA fragments by T4 DNA ligase indicates that protein MC1 mediates DNA bending. Several negatively supercoiled topoisomers of minicircles were obtained with DNA fragments of 203 and 146 bp, their distribution depends upon the amount of protein MC1 complexed with DNA. In addition, protein MC1 can induce a compaction of a nicked plasmid.
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PMID:The chromosomal protein MC1 from the archaebacterium Methanosarcina sp. CHTI 55 induces DNA bending and supercoiling. 205 61

Human cDNA clones encoding the major DNA ligase activity in proliferating cells, DNA ligase I, were isolated by two independent methods. In one approach, a human cDNA library was screened by hybridization with oligonucleotides deduced from partial amino acid sequence of purified bovine DNA ligase I. In an alternative approach, a human cDNA library was screened for functional expression of a polypeptide able to complement a cdc9 temperature-sensitive DNA ligase mutant of Saccharomyces cerevisiae. The sequence of an apparently full-length cDNA encodes a 102-kDa protein, indistinguishable in size from authentic human DNA ligase I. The deduced amino acid sequence of the human DNA ligase I cDNA is 40% homologous to the smaller DNA ligases of S. cerevisiae and Schizosaccharomyces pombe, homology being confined to the carboxyl-terminal regions of the respective proteins. Hybridization between the cloned sequences and mRNA and genomic DNA indicates that the human enzyme is transcribed from a single-copy gene on chromosome 19.
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PMID:Human DNA ligase I cDNA: cloning and functional expression in Saccharomyces cerevisiae. 220 63

Biochemical and genetic analyses have been conducted to determine whether a vaccinia virus open reading frame (orf) with extensive homology to the Saccharomyces cerevisiae DNA ligase gene encodes a functional ligase activity. This orf in HindIII A, designated A50R, is capable of encoding a 552-amino-acid, 63.4-kDa polypeptide. Full-length A50R mRNA produced in vitro directed the synthesis of a polypeptide with an apparent molecular weight of 57 kDa. Significantly, translation reactions programmed with A50R mRNA were capable of ligating a 3-kb Notl restriction fragment into multimers. DNA ligase activity was not detectable when either truncated sense or full-length antisense mRNA was translated in vitro. In extracts prepared from cells infected with wt vaccinia virus, DNA ligase activity was detected as assayed by the formation of a 57 kDa ligase-AMP adduct which was expressed early in the viral replication cycle. In cells infected with a DNA ligase deletion mutant no equivalent AMP-labeled adduct was detected. Relative to wt virus, the DNA ligase deletion mutant exhibited no significant differences in homologous recombination. These results indicate that the vaccinia orf A50R encodes a functional DNA ligase expressed early in infection, but this DNA ligase is nonessential for either recombination or viral replication.
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PMID:A DNA ligase gene in the Copenhagen strain of vaccinia virus is nonessential for viral replication and recombination. 221 23

A complex network of interacting proteins and enzymes is required for DNA replication. Much of our present understanding is derived from studies of the bacterium Escherichia coli and its bacteriophages T4 and T7. These results served as a guideline for the search and the purification of analogous proteins in eukaryotes. model systems for replication, such as the simian virus 40 DNA, lead the way. Generally, DNA replication follows a multistep enzymatic pathway. Separation of the double-helical DNA is performed by DNA helicases. Synthesis of the two daughter strands is conducted by two different DNA polymerases: the leading strand is replicated continuously by DNA polymerase delta and the lagging strand discontinuously in small pieces by DNA polymerase alpha. The latter is complexed to DNA primase, an enzyme in charge of frequent RNA primer syntheses on the lagging strand. Both DNA polymerases require several auxiliary proteins. They appear to make the DNA polymerases processive and to coordinate their functional tasks at the replication fork. 3'----5'-exonuclease, mostly part of the DNA polymerase delta polypeptide, can perform proof-reading by excising incorrectly base-paired nucleotides. The short DNA pieces of the lagging strand, called Okazaki fragments, are processed to a long DNA chain by the combined action of RNase H and 5'----3'-exonuclease, removing the RNA primers, DNA polymerase alpha or beta, filling the gap, and DNA ligase, sealing DNA pieces by phosphodiester bond formation. Torsional stress during DNA replication is released by DNA topoisomerases. In contrast to prokaryotes, DNA replication in eukaryotes not only has to create two identical daughter strands but also must conserve higher-order structures like chromatin.
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PMID:Eukaryotic DNA replication. Enzymes and proteins acting at the fork. 226 94

The differential ability of mammalian DNA ligases to use oligo(dT).poly(rA) as a substrate has been used to detect, and thereby extensively purify, two immunologically distinct forms of DNA ligase from rat liver. The activity of DNA ligase I, which is unable to use this template, is uniquely increased during liver regeneration, while that of DNA ligase II remains at a low level. Both enzymes require ATP and Mg2+ for activity and form an adenylylated intermediate which is stable and reactive. After SDS-PAGE, such radiolabeled complexes correspond to polypeptides of 130,000 and 80,000 Da for DNA ligase I and to 100,000 Da for DNA ligase II. That these labeled polypeptides do indeed correspond to active polypeptides of two different forms of DNA ligase is shown by the removal of the radiolabeled AMP, only when the intermediate is incubated with an appropriate substrate. In contrast to other eukaryotic DNA ligases, rat liver DNA ligase II has a lower Km for ATP (1.2 X 10(-5) M) than DNA ligase I (6 X 10(-5) M). Also, DNA ligase II can use ATP alpha S as a cofactor in the ligation reaction much more efficiently than DNA ligase I, further discriminating the ATP binding sites of these enzymes. Finally, antibodies raised against the 130,000-Da polypeptide of DNA ligase I specifically recognize this species in an immunoblot and inhibit only the activity of DNA ligase I.
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PMID:DNA ligases from rat liver. Purification and partial characterization of two molecular forms. 238 69

Vaccinia virus gene SalF 15R potentially encodes a polypeptide of 63 kD which shares 30% amino acid identity with S. pombe and S. cerevisiae DNA ligases. DNA ligase proteins can be identified by incubation with alpha-(32P)ATP, resulting in the formation of a covalent DNA ligase-AMP adduct, an intermediate in the enzyme reaction. A novel radio-labelled polypeptide of approximately 61 kD appears in extracts from vaccinia virus infected cells after incubation with alpha-(32P)ATP. This protein is present throughout infection and is a DNA ligase as the radioactivity is discharged in the presence of either DNA substrate or pyrophosphate. DNA ligase assays show an increase in enzyme activity in cell extracts after vaccinia virus infection. A rabbit antiserum, raised against a bacterial fusion protein of beta-galactosidase and a portion of SalF 15R, immune-precipitates polypeptides of 61 and 54 kD from extracts of vaccinia virus-infected cells. This antiserum also immune-precipitates the novel DNA ligase-AMP adduct, thus proving that the observed DNA ligase is encoded by SalF 15R.
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PMID:Vaccinia virus encodes a polypeptide with DNA ligase activity. 258 53

DNA ligase activity was studied in several untransformed or virus-transformed human cell lines from normal donors and from Bloom's syndrome (BS) patients. This proneness genetic disease is characterized by several cytological abnormalities and cancer proneness and, recently, some transformed cell lines from these patients were described to present a reduced activity of DNA ligase I. Results presented in this work indicate that: (i) the total DNA ligase activity in crude extract from untransformed or transformed cell lines from several BS patients was significantly higher than in control cells; (ii) the partial purification of the enzyme after gel filtration on fast protein liquid chromatography of crude extracts from lymphoblastoid BS cells showed that the enzyme activity was eluted in a major 180 kDa form in which activity was higher than in control cells; (iii) the activity gel analysis of these enzyme fractions revealed that DNA ligase of human cells was correlated to a major 130 kDa polypeptide and, in BS cells, the extent of the activity of this band was equal or higher than that in control untransformed or transformed cells.
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PMID:DNA ligase activity in human cell lines from normal donors and Bloom's syndrome patients. 272 53

DNA ligase II has been purified about 4,000-fold to apparent homogeneity from a calf thymus extract. The ligase consists of a single polypeptide with a molecular weight of 68,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On fluorography after electrophoresis, a DNA ligase-[3H]AMP complex gave a single band corresponding to a molecular weight of 68,000. The Km values of the ligase for ATP and nicked DNA (5'-phosphoryl ends) were obtained to be 40 and 0.04 microM, respectively. Antibody against calf thymus DNA ligase II was prepared by injecting the purified enzyme into a rabbit. The antibody cross-reacted with DNA ligase II but not with calf thymus DNA ligase I. DNA ligase II was not affected by antibody against calf thymus DNA ligase I with a molecular weight of 130,000 (Teraoka, H. and Tsukada, K. (1982) J. Biol. Chem. 257, 4758-4763). These results indicate that DNA ligase II (Mr = 68,000) is immunologically distinct from DNA ligase I (Mr = 130,000).
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PMID:Purification of DNA ligase II from calf thymus and preparation of rabbit antibody against calf thymus DNA ligase II. 308 91

Coliphage N4 replication is independent of most host DNA replication functions except for the 5'----3' exonuclease activity of polA, DNA ligase, DNA gyrase, and ribonucleotide reductase (Guinta, D., Stambouly, J., Falco, S. C., Rist, J. K., and Rothman-Denes, L. B. (1986) Virology 150, 33-44). It is therefore expected that N4 codes for most of the functions required for replication of its genome. In this paper we report the purification of the N4-coded DNA polymerase from N4-infected cell extracts by following its activity on a gapped template and in an in vitro complementation system for N4 DNA replication (Rist, J. K., Pearle, M., Sugino, A., and Rothman-Denes, L. B. (1986) J. Biol. Chem. 261, 10506-10510). The enzyme is composed of one polypeptide, Mr 87,000. It is most active on templates containing short gaps synthesizing DNA with high fidelity in a quasi-processive manner. A strong 3'----5' exonuclease activity is associated with the DNA polymerase polypeptide. No 5'----3' exonuclease or strand-displacing activities were detected.
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PMID:Purification and characterization of bacteriophage N4-induced DNA polymerase. 340 28

We have developed conditions for efficient cDNA cloning of nanogram amounts of purified mRNAs coding for cystathionine beta-synthase [L-serine hydro-lyase (adding homocysteine), EC 4.2.1.22] and for the cytosolic precursors of mitochondrial ornithine transcarbamylase (carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) and the beta subunit of propionyl-CoA carboxylase [propanoyl-CoA: carbon-dioxide ligase (ADP-forming), EC 6.4.1.3]. The three mRNAs, prepared by sequential immunoselection from the same batch of rat liver polysomes, were pooled (20 ng each), and cDNA was synthesized by using avian reverse transcriptase. The second DNA strand was prepared by "nick-translation repair" of the cDNA . mRNA hybrid with RNase H, polymerase I, and DNA ligase from Escherichia coli. The double-stranded (ds) DNA was tailed with deoxycytidine residues, annealed with Pst I-cut/dG-tailed pBR322, and used to transform E. coli. The library generated by this three-step procedure contained 5000 independent colonies. A 550-base-pair (bp) cDNA clone of the beta subunit of propionyl-CoA carboxylase was detected by hybrid-selected translation; it was then used to screen the library for longer cDNAs. Two hybridizing cDNAs, 1200 and 1000 bp long with a 200-bp overlap, representing together a full-length copy of the coding region and 446 bp of 3' untranslated sequence, were recovered. Each plasmid mapped to the region q13.3----q22 of human chromosome 3. Cystathionine beta-synthase clones were obtained by screening the library with a single-stranded [32P]cDNA prepared directly from the highly purified synthase mRNA by reverse transcriptase. The longest hybridizing cDNA of 1700 bp was used in hybrid-selected translation and detected a polypeptide of 63 kDa, identical in size to rat liver synthase. In situ hybridization of this cDNA to q22 of human chromosome 21 confirmed two previous tentative assignments of the synthase locus to this chromosome.
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PMID:Cloning and screening with nanogram amounts of immunopurified mRNAs: cDNA cloning and chromosomal mapping of cystathionine beta-synthase and the beta subunit of propionyl-CoA carboxylase. 345 73


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