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:5.99.1.2 (topoisomerase)
9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Transcriptional analysis by Northern blotting identified clusters of early, middle and late transcribed regions of the temperate lactococcal bacteriophage TP901-1 during one-step growth experiments. The latent period was found to be 65 min and the burst size 40 +/- 10. The eight early transcripts, all mapping in a 13 kb region adjacent to the attachment site of TP901-1, were present at maximal levels 10 min after infection. The four middle transcripts, observed at maximal levels 30 min after infection, are all located within a 2 kb region at the distal end of the early transcripts. The late class of transcripts were detected 40 min after infection and the amounts of these transcripts increased with time. The late transcripts were localized to the 13 kb region adjacent to the 2 kb middle transcribed region. The sequence of almost 4 kb of the early region was determined, allowing a detailed transcriptional map for the early region of which in total 6.4 kb was sequenced. Sequence analysis of the early region revealed two closely positioned but divergently orientated promoters, PL and PR, in accordance with the orientation of the ORFs and the transcriptional map. Nine ORFs were found, and similarities to a phage repressor, a single-stranded DNA-binding protein, a topoisomerase, a Cro-like protein and two other phage proteins of unknown function were detected. The gene arrangement in the early transcribed region of TP901-1 thus consists of two transcriptional units: one from PR containing four genes, of which at least two (the integrase gene and putative repressor) are needed for lysogeny, and the divergent and longer transcriptional unit from PL, presumably encoding functions required for the lytic life cycle. ORFs with homology to proteins involved in DNA replication were identified on the latter transcriptional unit.
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PMID:Temporal transcription of the lactococcal temperate phage TP901-1 and DNA sequence of the early promoter region. 972 42

Integration of the human immunodeficiency virus type 1 (HIV-1) cDNA is a required step for viral replication. Integrase, the virus-encoded enzyme important for integration, has not yet been exploited as a target for clinically useful inhibitors. Here we report on the identification of new polyhydroxylated aromatic inhibitors of integrase including ellagic acid, purpurogallin, 4,8, 12-trioxatricornan, and hypericin, the last of which is known to inhibit viral replication. These compounds and others were characterized in assays with subviral preintegration complexes (PICs) isolated from HIV-1-infected cells. Hypericin was found to inhibit PIC assays, while the other compounds tested were inactive. Counterscreening of these and other integrase inhibitors against additional DNA-modifying enzymes revealed that none of the polyhydroxylated aromatic compounds are active against enzymes that do not require metals (methylases, a pox virus topoisomerase). However, all were cross-reactive with metal-requiring enzymes (restriction enzymes, a reverse transcriptase), implicating metal atoms in the inhibitory mechanism. In mechanistic studies, we localized binding of some inhibitors to the catalytic domain of integrase by assaying competition of binding by labeled nucleotides. These findings help elucidate the mechanism of action of the polyhydroxylated aromatic inhibitors and provide practical guidance for further inhibitor development.
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PMID:Human immunodeficiency virus type 1 cDNA integration: new aromatic hydroxylated inhibitors and studies of the inhibition mechanism. 973 43

A series of new hydroxybenzoic and hydroxycinnamic acid flavon-3-yl esters were synthesized in order to obtain compounds targeting the human immunodeficiency virus (HIV) type 1 integrase (IN). The esters were tested for anti-IN and anti-reverse transcriptase (RT) activity in enzyme assays and for anti-HIV-1, anti-proliferative and anti-topoisomerase activity in cell-based assays. In enzyme assays, the two gallic acid flavon-3-yl esters showed a notable IN inhibition (IC50 values were 8.3 and 9.1 microM, respectively), while the two caffeic acid flavon-3-yl esters exhibited a modest activity (IC50 75 and 60 microM, respectively). Replacement of hydroxyl groups resulted in loss of potency. Caffeic acid 3',4'-dichloroflavon-3-yl ester also inhibited the RT activity whereas it was not active on human topoisomerases. It therefore represents an interesting example of a compound specifically targeting more than one step of the virus replication cycle.
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PMID:Synthesis and anti-human immunodeficiency virus type 1 integrase activity of hydroxybenzoic and hydroxycinnamic acid flavon-3-yl esters. 986 88

The integrase family of site-specific recombinases catalyzes conservative rearrangements between defined segments of DNA. A highly conserved tetrad (RHRY) of catalytic residues is essential for this process. This tetrad is dispersed in two motifs in the linear sequence, but is configured appropriately in the catalytic pocket to execute the strand cleavage and rejoining reactions. A third conserved motif has been identified in the Xer subgroup of the integrase family. Mutational analysis of 12 conserved residues in this motif in the XerD protein from Salmonella typhimurium led to the identification of an essential fifth catalytic residue (lysine 172) which is implicated in strand cleavage or exchange. This lysine residue occupies part of the turn of an antiparallel beta-hairpin which forms one side of the catalytic cleft in XerD, and is found at similar positions among evolutionarily diverse integrase family members. Related antiparallel beta-hairpins are present in eucaryotic type IB topoisomerase enzymes which also contain a critical lysine residue in the turn of the hairpin. In both the integrase family and eucaryotic type IB topoisomerases, the catalytic lysine residues are in close contact with the substrates and may play similar roles in influencing the reactivity of the phosphotyrosine intermediates formed during reactions catalyzed by both enzymes.
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PMID:A newly identified, essential catalytic residue in a critical secondary structure element in the integrase family of site-specific recombinases is conserved in a similar element in eucaryotic type IB topoisomerases. 1035 26

DNA supercoiling is essential for bacterial cell survival. We demonstrated that DNA topoisomerase IV, acting in concert with topoisomerase I and gyrase, makes an important contribution to the steady-state level of supercoiling in Escherichia coli. Following inhibition of gyrase, topoisomerase IV alone relaxed plasmid DNA to a final supercoiling density (sigma) of -0.015 at an initial rate of 0.8 links min(-1). Topoisomerase I relaxed DNA at a faster rate, 5 links min(-1), but only to a sigma of -0.05. Inhibition of topoisomerase IV in wild-type cells increased supercoiling to approximately the same level as in a mutant lacking topoisomerase I activity (to sigma = -0.08). The role of topoisomerase IV was revealed by two functional assays. Removal of both topoisomerase I and topoisomerase IV caused the DNA to become hyper-negatively supercoiled (sigma = -0.09), greatly stimulating transcription from the supercoiling sensitive leu-500 promoter and increasing the number of supercoils trapped by lambda integrase site-specific recombination.
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PMID:Roles of topoisomerases in maintaining steady-state DNA supercoiling in Escherichia coli. 1071 32

Many examples of enzymes that have lost their catalytic activity and perform other biological functions are known. The opposite situation is rare. A previously unnoticed structural similarity between the lambda integrase family (Int) proteins and the AraC family of transcriptional activators implies that the Int family evolved by duplication of an ancient DNA-binding homeodomain-like module, which acquired enzymatic activity. The two helix-turn-helix (HTH) motifs in Int proteins incorporate catalytic residues and participate in DNA binding. The active site of Int proteins, which include the type IB topoisomerases, is formed at the domain interface and the catalytic tyrosine residue is located in the second helix of the C-terminal HTH motif. Structural analysis of other 'tyrosine' DNA-breaking/rejoining enzymes with similar enzyme mechanisms, namely prokaryotic topoisomerase I, topoisomerase II and archaeal topoisomerase VI, reveals that the catalytic tyrosine is placed in a HTH domain as well. Surprisingly, the location of this tyrosine residue in the structure is not conserved, suggesting independent, parallel evolution leading to the same catalytic function by homologous HTH domains. The 'tyrosine' recombinases give a rare example of enzymes that evolved from ancient DNA-binding modules and present a unique case for homologous enzymatic domains with similar catalytic mechanisms but different locations of catalytic residues, which are placed at non-homologous sites.
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PMID:Two tricks in one bundle: helix-turn-helix gains enzymatic activity. 1087 43

The study of biochemical pathways requires the isolation and characterization of each and every intermediate in the pathway. For the site-specific recombination reactions catalyzed by the bacteriophage lambda tyrosine recombinase integrase (Int), this has been difficult because of the high level of efficiency of the reaction, the highly reversible nature of certain reaction steps, and the lack of requirements for high-energy cofactors or metals. By screening synthetic peptide combinatorial libraries, we have identified two related hexapeptides, KWWCRW and KWWWRW, that block the strand-cleavage activity of Int but not the assembly of higher-order intermediates. Although the peptides bind DNA, their inhibitory activity appears to be more specifically targeted to the Int-substrate complex, insofar as inhibition is resistant to high levels of non-specific competitor DNA and the peptides have higher levels of affinity for the Int-DNA substrate complex than for DNA alone. The peptides inhibit the four pathways of Int-mediated recombination with different potencies, suggesting that the interactions of the Int enzyme with its DNA substrates differs among pathways. The KWWCRW and KWWWRW peptides also inhibit vaccinia virus topoisomerase, a type IB enzyme, which is mechanistically and structurally related to Int. The peptides differentially affect the forward and reverse DNA transesterification steps of the vaccinia topoisomerase. They block formation of the covalent vaccinia topoisomerase-DNA intermediate, but have no apparent effect on DNA religation by preformed covalent complexes. The peptides also inhibit Escherichia coli topoisomerase I, a type IA enzyme. Finally, the peptides inhibit the bacteriophage T4 type II topoisomerase and several restriction enzymes with 2000-fold lower potency than they inhibit integrase in the bent-L pathway.
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PMID:Peptide inhibitors of DNA cleavage by tyrosine recombinases and topoisomerases. 1087 46

We have developed microtiter assays for detecting catalysis by type IB topoisomerases and retroviral integrases. Each assay employs model DNA substrates containing biotin in one strand and digoxigenin in another. In each case action of the enzyme results in the formation of a single DNA strand containing both groups. This allows the reaction product to be quantified by capturing biotinylated product DNA on avidin-coated plates followed by detection using an anti-digoxigenin ELISA. The order of addition of reactants and inhibitors can be varied to distinguish effects of test compounds on different steps in the reaction. These assays were used to screen compound libraries for inhibitors active against mammalian topoisomerase or HIV integrase. We identified (-)-epigallocatechin 3-O:-gallate, as a potent inhibitor of religation by mammalian topoisomerase (IC(50) of 26 nM), potentially explaining the anti-cancer properties previously attributed to this compound. New integrase inhibitors were also identified. A similar strategy may be used to develop microtiter assays for many further DNA modifying enzymes.
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PMID:Rapid microtiter assays for poxvirus topoisomerase, mammalian type IB topoisomerase and HIV-1 integrase: application to inhibitor isolation. 1112 79

Previously we have characterized type IB DNA topoisomerase V (topo V) in the hyperthermophile Methanopyrus kandleri. The enzyme has a powerful topoisomerase activity and is abundant in M. kandleri. Here we report two characterizations of topo V. First, we found that its N-terminal domain has sequence homology with both eukaryotic type IB topoisomerases and the integrase family of tyrosine recombinases. The C-terminal part of the sequence includes 12 repeats, each repeat consisting of two similar but distinct helix-hairpin-helix motifs; the same arrangement is seen in recombination protein RuvA and mammalian DNA polymerase beta. Second, on the basis of sequence homology between topo V and polymerase beta, we predict and demonstrate that topo V possesses apurinic/apyrimidinic (AP) site-processing activities that are important in base excision DNA repair: (i) it incises the phosphodiester backbone at the AP site, and (ii) at the AP endonuclease cleaved AP site, it removes the 5' 2-deoxyribose 5-phosphate moiety so that a single-nucleotide gap with a 3'-hydroxyl and 5'-phosphate can be filled by a DNA polymerase. Topo V is thus the prototype for a new subfamily of type IB topoisomerases and is the first example of a topoisomerase with associated DNA repair activities.
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PMID:A type IB topoisomerase with DNA repair activities. 1135 38

The tyrosine family site-specific recombinases, in contrast to the related type I topoisomerases, which act as monomers on a single DNA molecule, rely on multi-protein complexes to synapse partner DNAs and coordinate two sequential strand exchanges involving four nicking-closing reactions. Here, we analyze three mutants of the catalytic domain of lambda integrase (Int), A241V, I353M and W350ter that are defective for normal recombination, but possess increased topoisomerase activity. The mutant enzymes can carry out individual DNA strand exchanges using truncated substrates or Holliday junctions, and they show more DNA-cleavage activity than wild-type Int on isolated att sites. Structural modeling predicts that the substituted residues may destabilize interactions between the C-terminal beta-strand (beta7) of Int and the core of the protein. The cleavage-competent state of Int requires the repositioning of the nucleophile (Y342) located on beta6 and the catalyst K235 located on the flexible beta2-beta3 loop, relative to their positions in a crystal structure of the inactive conformation. We propose that the anchoring of beta7 against the protein core restrains the movement of Tyr342 and/or Lys235, causing an attenuation of cleavage activity in most contexts. Within a bona fide recombination complex, the release of strand beta7 would allow Tyr342 and Lys235 to assume catalytically active conformations in coordination with other Int protomers in the complex. The loss of beta7 packing by misalignment or truncation in the mutant proteins described here causes a loss of regulated activity, thereby favoring DNA cleavage activity in monomeric complexes and forfeiting the coordination of strand-exchange necessary for efficient recombination.
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PMID:Attenuating functions of the C terminus of lambda integrase. 1246 May 68


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