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

We have initiated the characterization of the DNA helicases from HeLa cells, and we have observed at least 4 molecular species as judged by their different fractionation properties. One of these only, DNA helicase I, has been purified to homogeneity and characterized. Helicase activity was measured by assaying the unwinding of a radioactively labelled oligodeoxynucleotide (17 mer) annealed to M13 DNA. The apparent molecular weight of helicase I on SDS polyacrylamide gel electrophoresis is 65 kDa. Helicase I reaction requires a divalent cation for activity (Mg2+ greater than Mn2+ greater than Ca2+) and is dependent on hydrolysis of ATP or dATP. CTP, GTP, UTP, dCTP, dGTP, dTTP, ADP, AMP and non-hydrolyzable ATP analogues such as ATP gamma S are unable to sustain helicase activity. The helicase activity has an optimal pH range between pH8.0 to pH9.0, is stimulated by KCl or NaCl up to 200mM, is inhibited by potassium phosphate (100mM) and by EDTA (5mM), and is abolished by trypsin. The unwinding is also inhibited competitively by the coaddition of single stranded DNA. The purified fraction was free of DNA topoisomerase, DNA ligase and nuclease activities. The direction of unwinding reaction is 3' to 5' with respect to the strand of DNA on which the enzyme is bound. The enzyme also catalyses the ATP-dependent unwinding of a DNA:RNA hybrid consisting of a radioactively labelled single stranded oligodeoxynucleotide (18 mer) annealed on a longer RNA strand. The enzyme does not require a single stranded DNA tail on the displaced strand at the border of duplex regions; i.e. a replication fork-like structure is not required to perform DNA unwinding. The purification of the other helicases is in progress.
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PMID:A DNA helicase from human cells. 170 1

Administration of hepatocarcinogens aflataxin B1 (AFB1) and N-nitrosodimethylamine (NDMA) to rats caused single-strand breaks in hepatic nuclear DNA. The damage was found to be maximum at 4 hours following AFB1 administration and at 2 hours following NDMA administration. These damages were repaired after 17 and 4 hours, respectively in cases of AFB1 and NDMA. The activity of poly(ADP-ribose)polymerase (PARP), an enzyme known to use single-strand breaks of DNA as cofactor, was observed to increase with increasing damage to DNA and decrease as and when this damage got repaired. DNA polymerase beta and DNA ligase activities were also seen to increase and decline in a way analogous to PARP. In contrast, DNA topoisomerase activity declined corresponding to an increase in PARP activity. These observations suggest a possible role of PARP in coordinating the activities of other enzymes involved in DNA repair. It is also envisaged that these parameters can be utilized to devise strategies to counteract the deleterious effects of chemical carcinogens.
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PMID:Activity of some nuclear enzymes associated with DNA repair following hepatocarcinogen administration to rats. 759 30

Construction of chimaeric DNA molecules in vitro relies traditionally on two enzymatic steps catalyzed by separate protein components. Site-specific restriction endonucleases are used to generate linear DNAs with defined termini that can then be joined covalently at their ends via the action of DNA ligase. A novel approach to the synthesis of recombinant DNAs exploits the ability of a single enzyme, vaccinia DNA topoisomerase, to both cleave and rejoin DNA strands with extreme specificity at each step. Placement of the CCCTT cleavage motif for vaccinia topoisomerase near the end of a duplex DNA permits efficient generation of a stable, highly recombinogenic protein-DNA adduct that can religate only to acceptor DNAs that contain complementary single-strand extensions. Linear DNAs containing CCCTT cleavage sites at both ends (bivalent substrates) can be activated by topoisomerase and inserted into a plasmid vector in a simple and rapid in vitro procedure that is especially well suited to the molecular cloning of polymerase chain reaction-amplified DNAs. Activation of polyvalent (e.g. branched) DNA substrates by topoisomerase offers a potentially powerful method for the synthesis of two- and three-dimensional polynucleotide networks.
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PMID:Novel approach to molecular cloning and polynucleotide synthesis using vaccinia DNA topoisomerase. 779 75

The nucleotide sequence of a 55098 bp region from the right end of the genome of a virulent African swine fever virus (ASFV) isolate (Malawi LIL20/1) has been determined. Translation of the sequence identified 67 major open reading frames (ORFs) which are closely spaced and read from both DNA strands. At six positions intergenic tandem repeat arrays are found. Comparison of the predicted amino acid sequences of encoded proteins with protein sequence databases identified a number of homologies. These include three subunits of RNA polymerase, a protein with homology to transcription factor SII (TFSII), a DNA ligase, two subunits of mRNA capping enzyme, a DNA topoisomerase type II, a dUTPase, a protein kinase, three helicases, a ubiquitin-conjugating enzyme, a protein with homology to the nif S and nif S-like proteins identified in some bacteria and Saccharomyces cerevisiae, a protein with homology to both a myeloid differentiation primary response antigen (MyD116) and to a herpes simplex virus-encoded neurovirulence-associated protein (ICP34.5), a protein with homology to the ASFV-encoded structural protein p22, two proteins with homology to copies of the ASFV-encoded multigene family 360 and one protein with homology to the ASFV-encoded multigene family 110. Four genes encode proteins which have homology to each other and constitute a new multigene family (MGF100). Nine ORFs encode proteins which contain predicted transmembrane domains. The possible functions of these predicted ASFV-encoded proteins are discussed and the evolutionary relationship of ASFV to other viruses are considered. Despite the similarities in genome structure and replication strategy of ASFV with poxviruses, sequence similarity between them is low and the organization of ASFV-encoded genes is not colinear with that of the orthopoxviruses.
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PMID:Nucleotide sequence of a 55 kbp region from the right end of the genome of a pathogenic African swine fever virus isolate (Malawi LIL20/1). 802 96

An ATP-dependent DNA helicase has been purified to near homogeneity from pea chloroplasts. The enzyme is a homodimer of 68-kDa subunits. The purified enzyme shows DNA-dependent ATPase activity and is devoid of DNA polymerase, DNA topoisomerase, DNA ligase or nuclease activities. The enzyme requires Mg2+ or Mn2+ for its maximum activity. ATP is the most favoured cofactor for this enzyme while other NTP or dNTP are poorly utilized. Pea chloroplast DNA helicase can unwind a 17-bp duplex whether it has unpaired single-stranded tails at both the 5' end and 3' end, at the 5' end or at the 3' end only, or at neither end. However, it fails to act on a blunt-ended 17-bp duplex DNA. The enzyme moves unidirectionally from 3' to 5' along the bound strand. The unwinding activity is inhibited by the intercalating drugs nogalamycin and daunorubicine.
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PMID:Purification and characterization of a DNA helicase from pea chloroplast that translocates in the 3'-to-5' direction. 866 52

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

Polymerase chain reaction analysis of a large collection of bacteriophages with T-even morphology revealed four phages that are distantly related to all the others. The genomes of these pseudo T-even phages hybridized under stringent conditions to only a limited portion of the T4 genome that encodes virus head, head-to-tail joining and contractile tail genes. Except for this region, no extensive hybridization was detected between most pairs of the different pseudo T-even genomes. Sequencing of this conserved region of the pseudo T-even phage RB49 revealed substantial nucleotide sequence divergence from T4 (approximately 30% to 40%), and random genomic sequencing of this phage indicated that more than a third of its sequences had no detectable homology to T4. Among those sequences related to the T-even genes were virion structural components including the constituents of the phage base plate. Only a few sequences had homology to T4 early functions; these included ribonucleotide diphosphatase reductase, DNA ligase and the large subunit of DNA topoisomerase. The genomes of the pseudo T-even phage were digested by restriction enzymes that are unable to digest the T-even DNAs which contain glucosylated hydroxymethyl-cytosine residues. This suggests that only limited nucleotide modifications must be present in the pseudo T-even genomes. Conservation of much of the morphogenetic region of these diverse phage genomes may reflect particularly strong sequence constraints on these gene products. However, other explanations are considered, including the possibility that the various morphogenetic segments were acquired by the pseudo T-even genomes by modular evolution. These results support the notion that phage evolution may proceed within a network of both closely and distantly related genomes.
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PMID:The genome of the pseudo T-even bacteriophages, a diverse group that resembles T4. 909 22

Polycyclic aromatic hydrocarbon (PAH)-DNA adducts pervert the execution or fidelity of enzymatic DNA transactions and cause mutations and cancer. Here, we examine the effects of intercalating PAH-DNA adducts on the religation reaction of vaccinia DNA topoisomerase, a prototypal type IB topoisomerase (TopIB), and the 3' end-resection reaction of Escherichia coli exonuclease III (ExoIII), a DNA repair enzyme. Vaccinia TopIB forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a target site 5'-C(+5)C(+4)C(+3)T(+2)T(+1)p / N(-1) in duplex DNA. The rate of the forward cleavage reaction is suppressed to varying degrees by benzo[a]pyrene (BP) or benzo[c]phenanthrene (BPh) adducts at purine bases within the 3'-G(+5)G(+4)G(+3)A(+2)A(+1)T(-1)A(-2) sequence of the nonscissile strand. We report that BP adducts at the +1 and -2 N6-deoxyadenosine (dA) positions flanking the scissile phosphodiester slow the rate of DNA religation to a greater degree than they do the cleavage rate. By increasing the cleavage equilibrium constant > or = 10-fold, the BPdA adducts, which are intercalated via the major groove, act as TopIB poisons. With respect to ExoIII, we find that (i) single BPdA adducts act as durable roadblocks to ExoIII digestion, which is halted at sites 1 and 2 nucleotides prior to the modified base; (ii) single BPhdA adducts, which also intercalate via the major groove, elicit a transient pause prior to the lesion, which is eventually resected; and (iii) BPh adducts at N2-deoxyguanosine, which intercalate via the minor groove, are durable impediments to ExoIII digestion. These results highlight the sensitivity of repair outcomes to the structure of the PAH ring system and whether intercalation occurs via the major or minor groove.
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PMID:Intercalating polycyclic aromatic hydrocarbon-DNA adducts poison DNA religation by Vaccinia topoisomerase and act as roadblocks to digestion by exonuclease III. 1676 60

Plants and algae possess plastids and mitochondria harboring their own genomes, which are replicated by the apparatus consisting of DNA polymerase, DNA primase, DNA helicase, DNA topoisomerase, single-stranded DNA maintenance protein, DNA ligase, and primer removal enzyme. In the higher plant Arabidopsis thaliana, organellar replication-related enzymes (OREs) are similar in plastids and mitochondria because many of them are dually targeted to plastids and mitochondria. In the red algae, there is a report about a DNA replicase, plant/protist organellar DNA polymerase, which is localized to both plastids and mitochondria. However, other OREs remain unclear in algae. Here, we identified OREs possibly localized to organelles in the unicellular rhodophyte Cyanidioschyzon merolae. We then examined intracellular localization of green fluorescent protein-fusion proteins of these enzymes in C. merolae, whose cell has a single plastid and a single mitochondrion and is suitable for localization analysis, demonstrating that the plastid and the mitochondrion contain markedly different components of replication machinery. Phylogenetic analyses revealed that the organelle replication apparatus was composed of enzymes of various different origins, such as proteobacterial, cyanobacterial, and eukaryotic, in both red algae and green plants. Especially in the red alga, many enzymes of cyanobacterial origin remained. Finally, on the basis of the results of localization and phylogenetic analyses, we propose a model on the succession of OREs in eukaryotes.
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PMID:Localization and phylogenetic analysis of enzymes related to organellar genome replication in the unicellular rhodophyte Cyanidioschyzon merolae. 2440 55

Plastids and mitochondria possess their own genomes. Although the replication mechanisms of these organellar genomes remain unclear in photosynthetic eukaryotes, several organelle-localized enzymes related to genome replication, including DNA polymerase, DNA primase, DNA helicase, DNA topoisomerase, single-stranded DNA maintenance protein, DNA ligase, primer removal enzyme, and several DNA recombination-related enzymes, have been identified. In the reference Eudicot plant Arabidopsis thaliana, the replication-related enzymes of plastids and mitochondria are similar because many of them are dual targeted to both organelles, whereas in the red alga Cyanidioschyzon merolae, plastids and mitochondria contain different replication machinery components. The enzymes involved in organellar genome replication in green plants and red algae were derived from different origins, including proteobacterial, cyanobacterial, and eukaryotic lineages. In the present review, we summarize the available data for enzymes related to organellar genome replication in green plants and red algae. In addition, based on the type and distribution of replication enzymes in photosynthetic eukaryotes, we discuss the transitional history of replication enzymes in the organelles of plants.
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PMID:Enzymes involved in organellar DNA replication in photosynthetic eukaryotes. 2527 52


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