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)

The polynucleotide ligase-catalyzed joining of the eight chemically synthesized deoxypolynucleotides (segments 19 to 26), comprising the nucleotide sequence 86-126 of the DNA corresponding to the Escherichia coli tyrosine tRNA precursor has been investigated. Joining was studied using various combinations of 3, 4, or larger number of segments at a time. The extent of joining was in general low (0 to 40%) for the three-component as well as for the four-component systems. Joining of the five- and six- component systems was more satisfactory with yields from 25 to about 60%. The three duplexes [IVa] to [IVc]were prepared in single step reactions in yields of about 50% and were characterized. Duplex [IVd] could not be prepared in a single step reaction because of the failure of 5'-phosphorylated segment 26 to join to the rest of the duplex. Using a carefully annealed mixture of segments 24, 25, and phosphorylated segment 26, the joining of the latter to segment 24 could be realized in about 25% yield, much activated intermediate being concurrently present.
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PMID:Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 10. Enzymatic joining of chemically synthesized segments to form the DNA duplex corresponding to the nucleotide sequence 86-126. 17 57

In continuing the work on the total synthesis of the gene for an Escherichia coli tyrosine suppressor tRNA (accompanying papers) and as a part of a study of the mechanism of transcription of this gene, a 23-nucleotide unit-long DNA corresponding to the previously determined (Loewen, P., Sekiya, T., and Khorana, H. G. (1974) J. Biol. Chem. 249, 217) sequence has been synthesized. The synthesis was carried out by dividing the total duplex into the following five deoxyribooligonucleotide segments, all of which were chemically synthesized: (a) the undecanucleotide, d(A-G-T-G-A-T-G-G-T-G-G); (b)the undecanucleotide, d(T-C-A-C-T-T-T-C-A-A-A); (c) the undecanucleotide, d(G-G-A-C-T-T-T-T-G-A-A); (d) the dodecanucleotide, d(A-G-T-C-C-C-T-G-A-A-C-T); and (e) the heptanucleotide, d(A-G-T-T-C-A-G). All the five synthetic oligonucleotides were characterized by chromatographic and radioactive fingerprinting methods after labeling the 5'-ends with a 32P-phosphate group. Synthesis of the double-stranded DNA duplex was completed by joining 5'-phosphorylated segments 1, 3, and 4 in the presence of segments 2 and 5 using T4-polynucleotide ligase. The DNA duplex was characterized.
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PMID:Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 12. Synthesis of a DNA duplex corresponding to a sequence of 23 nucleotide units adjoining the C-C-A end. 17 59

The method developed for the total synthesis of a given DNA containing biologically specific sequences consists of the following. The DNA in the double-stranded form is carefully divided into short single-stranded segments with suitable overlaps in the complementary strands. All the segments are chemically synthesized starting with protected nucleosides and mononucleotides. The 5'-OH ends of the appropriate oligonucleotides are then phosphorylated with the use of [y-32P]ATP and polynucleotide kinase. A few to several neighboring oligonucleotides are then allowed to form bihelical complexes in aqueous solution, and the latter are joined end to end by polynucleotide ligase to form covalently linked duplexes. Subsequent heat-to-tail joining of the short duplexes leads to the total DNA. The methods are described for the construction of a biologically functional suppressor transfer RNA gene. The total work involved (i) the synthesis of a 126-nucleotide-long bihelical DNA corresponding to a known precursor to the tyrosine suppressor transfer RNA, (ii) the sequencing of the promoter region and the distal region adjoining the C-C-A end, which contained a signal for the processing of the RNA transcript, (iii) total synthesis of the 207 base-pair-long DNA, which included the control elements, as well as the Eco R1 restriction endonuclease specific sequences at the two ends, and (iv) full characterization by transcription in vitro and amber suppressor activity in vivo of the synthetic gene.
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PMID:Total synthesis of a gene. 36 49

With the ultimate objective of the total synthesis of a tRNA gene including its transcriptional signals, an Escherichia coli tyrosine suppressor tRNA gene was chosen. The arguments in favor of this choice are presented. A plan for the total synthesis of the 126-nucleotide-long DNA duplex corresponding to a precursor (Altman S., and Smith, J. D. (1971) Nature New Biol. 233, 35) to the above tRNA is formulated. The plan involves: (a) the chemical synthesis of 26 deoxyribooligonucleotide segments, (b) polynucleotide ligase-catalyzed joining of several segments at a time to form a total of four DNA duplexes with appropriate comlementary single-stranded ends, and (c) the joining of the duplexes to form the entire DNA duplex. Ten accompanying papers describe the experimental realization of this objective.
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PMID:Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 1. General introduction. 76 27

Polynucleotide ligase-catalyzed joining of the eight chemically synthesized deoxyribopolynucleotide segments (Fig. 1) comprising the nucleotide sequence 23-66 of the DNA corresponding to the Escherichia coli tyrosine tRNA precursor has been systematically investigated. Joining was studied using all possible combinations of 3, 4, and 5 and larger numbers of segments at a time. The extent of joining varied widely (0 to about 90%) in three component systems. The "self-structure" of some of the components evidently inhibited the joining. Addition of a fourth segment in general enhanced the extent of joining and optimal yields were obtained in systems containing six or more segments. A comparison of the T4-induced ligase and the E. coli polynucleotide ligase for joining of the chemically synthesized segments showed the E. coli enzyme to be inferior to the T4-induced ligase. Satisfactory syntheses of the duplexes [IIa] and [IIb] comprising, respectively, eight and seven segments were achieved in single steps. Of the two terminal segments carrying 5'-OH groups in the duplexes, only one (segment 7) was used in the prephosphorylated form. The duplexes were isolated pure and characterized by enzymatic degradations and by electrophoresis.
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PMID:Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 8. Enzymatic joining of the chemically synthesized segments to form DNA duplexes corresponding to nucleotide sequences 23-60 and 23-66. 76 33

Previous structure/function analyses of the DNA repair enzyme, T4 endonuclease V, have suggested that the extreme carboxyl portion of the enzyme is associated with pyrimidine dimer-specific binding (Recinos and Lloyd, and Stump and Lloyd, Biochemistry 27:1832-1838 and 1839-1843, 1988, respectively). Within the final 11 amino acids there are 5 aromatic, 2 basic, and no acidic residues and it has been proposed that these residues stack with and electrostatically interact with the kinked DNA at the site of a pyrimidine dimer. The role of the tyrosine residue at position 129 has been investigated by oligonucleotide site-directed mutagenesis in which the codon for Tyr-129 has been altered to reflect conservative changes of Trp and Phe and more dramatic changes of Ser, a stop codon, deletion of the codon or introduction of a frameshift. Both changes to the aromatic amino acids resulted in proteins which accumulated well in E. coli and not only significantly enhanced the UV survival of repair-deficient cells but also complemented a defective denV gene within UV-irradiated T4 phage. Partially purified preparations of the Tyr-129----Trp and Tyr-129----Phe mutants were assayed for their ability to processively incise UV-irradiated plasmid DNA (a nicking reaction carried out at low 25 mM salt concentrations). The mutant enzymes Tyr-129----Phe and Tyr-129----Trp displayed a 1000% and 500% enhanced specific nicking activity, respectively. These reactions were also shown to be completely processive. Assays performed at higher (100 mM) salt concentrations reduced the specific activities of the mutant enzymes approximately to that of wild type for the Tyr-129----Phe mutant and to 20% that of wild type for the Tyr-129----Trp mutant.
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PMID:Site-directed mutagenesis of the T4 endonuclease V gene: mutations which enhance enzyme specific activity at low salt concentrations. 269 26

tRNA-guanine transglycosylase (Tgt) is involved in the biosynthesis of the hypermodified tRNA nucleoside queuosine (Q). It catalyzes the posttranscriptional base exchange of the Q precursor 7-aminomethyl-7-deazaguanine (preQ1) with the genetically encoded guanine in the anticodon of tRNA(Asp), tRNA(Asn), tRNA(His), and tRNA(Tyr). A partially sequenced gene upstream of the DNA ligase (lig) gene of the Zymomonas mobilis chromosome shows strong homology to the tgt gene of Escherichia coli (K.B. Shark and T. Conway, FEMS Microbiol. Lett. 96:19-26, 1992). We showed that this gene is able to complement the tgt mutation in E. coli SJ1505, and we determined its complete sequence. Four start codons were possible for this gene, resulting in proteins of 386 to 399 amino acids (M(r), 42,800 to 44,300) showing 60.4% sequence identity with Tgt from E. coli. The smallest of the four possible reading frames, which was still extended at its 5' end compared with the E. coli tgt gene, was overexpressed in E. coli. The gene product was purified to homogeneity and was biochemically characterized. The kinetical parameters were virtually identical to those published for the E. coli enzyme. In contrast to E. coli Tgt, which is reported to be a homotrimer, Z. mobilis Tgt was found to be a monomer according to gel filtration. In this study, it was shown that the formation of homotrimers by the E. coli enzyme is readily reversible and is dependent on protein concentration.
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PMID:Sequence analysis and overexpression of the Zymomonas mobilis tgt gene encoding tRNA-guanine transglycosylase: purification and biochemical characterization of the enzyme. 766 16

DNA topoisomerase I (topo I) is a member of a group of essential nuclear enzymes which control and modify the topological state of DNA and is recognized as the target for anticancer drugs. During the course of the catalytic activity of topo I, a covalent bond is formed between a tyrosine group at the active site of the enzyme and a 3' phosphate group along the DNA backbone. This chemical reaction resembles the protein kinase-mediated tyrosine phosphorylation process. We assumed, therefore, that tyrphostins, potent and selective blockers of protein tyrosine kinases, might affect topo I activity. We found that of three derivatives of tyrphostins (AG-555, AG-18, and AG-213) that inhibited topo I activity in an in vitro assay, AG-555 was the most active. Examination of the mechanism by which these compounds act as topo I inhibitors revealed that AG-555 blocked the binding of this enzyme to the DNA due to its interaction with the topo I enzyme. We showed that its mode of action differed from that observed for camptothecin, a known topo I inhibitor. However, AG-555 did not affect the activity of other major DNA binding enzymes (i.e., DNA ligase, DNA polymerase I, and reverse transcriptase). This study suggests that tyrphostins may serve as a new class of topo I inhibitors, and these results also present additional explanations for their antiproliferative effect.
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PMID:Inhibition of topoisomerase I activity by tyrphostin derivatives, protein tyrosine kinase blockers: mechanism of action. 792 31

Mammalian cells contain three biochemically distinct DNA ligases. In this report we describe the purification of DNA ligase II to homogeneity from bovine liver nuclei. This enzyme interacts with ATP to form an enzyme-AMP complex, in which the AMP moiety is covalently linked to a lysine residue. An adenylylated peptide from DNA ligase II contains the sequence, Lys-Tyr-Asp-Gly-Glu-Arg, which is homologous to the active site motif conserved in ATP-dependent DNA ligases. The sequences adjacent to this motif in DNA ligase II are different from the comparable sequences in DNA ligase I, demonstrating that these enzymes are encoded by separate genes. The amino acid sequences of 15 DNA ligase II peptides exhibit striking homology (65% overall identity) with vaccinia DNA ligase. These peptides are also homologous (31% overall identity) with the catalytic domain of mammalian DNA ligase I, indicating that the genes encoding DNA ligases I and II probably evolved from a common ancestral gene. Since vaccinia DNA ligase is not required for DNA replication but influences the ability of the virus to survive DNA damage, the homology between this enzyme and DNA ligase II suggests that DNA ligase II may be involved in DNA repair.
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PMID:Mammalian DNA ligase II is highly homologous with vaccinia DNA ligase. Identification of the DNA ligase II active site for enzyme-adenylate formation. 798 68

Interleukin-1beta-converting enzyme (ICE)/Ced-3 proteases play a critical role in apoptosis. One well characterized substrate of these proteases is the DNA repair enzyme poly(ADP-ribose) polymerase. We report here that alpha-fodrin, an abundant membrane-associated cytoskeletal protein, is cleaved rapidly and specifically during Fas- and tumor necrosis factor-induced apoptosis; this cleavage is mediated by an ICE/Ced-3 protease distinct from the poly(ADP-ribose) polymerase protease. Studies in cells treated with these apoptotic stimuli reveal that both fodrin and poly(ADP-ribose) polymerase proteolysis are inhibited by acetyl-Tyr-Val-Ala-Asp chloromethyl ketone and CrmA, specific inhibitors of ICE/Ced-3 proteases. However, fodrin proteolysis can be distinguished from poly(ADP-ribose) polymerase proteolysis by its relative insensitivity to acetyl-Asp-Glu-Val-Asp aldehyde (DEVD-CHO), a selective inhibitor of a subset of ICE/Ced-3 proteases that includes CPP32. DEVD-CHO protects cells from Fas-induced apoptosis but does not prevent fodrin proteolysis, indicating that cleavage of this protein can be uncoupled from apoptotic cell death. Moreover, purified fodrin is cleaved in vitro by CPP32 (but not by ICE) into fragments of the same size observed in vivo during apoptosis. These findings suggest that fodrin proteolysis in vivo may reflect the activity of multiple ICE/Ced-3 proteases whose partial sensitivity to DEVD-CHO reflects a limited contribution from CPP32, or an ICE/Ced-3 protease less sensitive than CPP32 to DEVD-CHO inhibition.
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PMID:Specific cleavage of alpha-fodrin during Fas- and tumor necrosis factor-induced apoptosis is mediated by an interleukin-1beta-converting enzyme/Ced-3 protease distinct from the poly(ADP-ribose) polymerase protease. 894 Jan 32


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