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At least 451 records · Page 25Linked to original sources
PubMed1999

The topological mechanism of phage lambda integrase.

Bacteriophage lambda integrase (Int) is a versatile site-specific recombinase. In concert with other proteins, it mediates phage integration into and excision out of the bacterial chromosome. Int recombines intramolecular sites in inverse or direct orientation or sites on separate DNA molecules. This wide spectrum of Int-mediated reactions has, however, hindered our understanding of the topology of Int recombination. By systematically analyzing the topology of Int reaction products and using a mathematical method called tangles, we deduce a unified model for Int recombination. We find that, even in the absence of (-) supercoiling, all Int reactions are chiral, producing one of two possible enantiomers of each product. We propose that this chirality reflects a right-handed DNA crossing within or between recombination sites in the synaptic complex that favors formation of right-handed Holliday junction intermediates. We demonstrate that the change in linking number associated with excisive inversion with relaxed DNA is equally +2 and -2, reflecting two different substrates with different topology but the same chirality. Additionally, we deduce that integrative Int recombination differs from excisive recombination only by additional plectonemic (-) DNA crossings in the synaptic complex: two with supercoiled substrates and one with relaxed substrates. The generality of our results is indicated by our finding that two other members of the integrase superfamily of recombinases, Flp of yeast and Cre of phage P1, show the same intrinsic chirality as lambda Int.

Bacteriophage P1↗
PubMed2000

Site-specific recombination in human cells catalyzed by phage lambda integrase mutants.

Phage lambda Integrase (Int) is the prototype of the so-called integrase family of conservative site-specific recombinases, which includes Cre and FLP. The natural function of Int is to execute integration and excision of the phage into and out of the Escherichia coli genome, respectively. In contrast to Cre and FLP, however, wild-type Int requires accessory proteins and DNA supercoiling of target sites to catalyze recombination. Here, we show that two mutant Int proteins, Int-h (E174 K) and its derivative Int-h/218 (E174 K/E218 K), which do not require accessory factors, are proficient to perform intramolecular integrative and excisive recombination in co-transfection assays inside human cells. Intramolecular integrative recombination is also detectable by Southern analysis in human reporter cell lines harboring target sites attB and attP as stable genomic sequences. Recombination by wild-type Int, however, is not detectable by this method. The latter result implies that eukaryotic co-factors, which could functionally replace the prokaryotic ones normally required for wild-type Int, are most likely not present in human cells.

Attachment Sites, Microbiological↗
PubMed1996

Biochemical characterization of recombinant equine infectious anemia virus integrase.

The integrase from equine infectious anemia virus (ELAV) was expressed in Escherichia coli as a polyhistidine fusion protein. The protein was purified under native and denaturing conditions using one-step nickel-affinity chromatography. The purified denatured protein was refolded in the presence of detergent. In vitro 3' processing and DNA strand transfer activities were analyzed under Mg(2+)- and Mn(2+)-dependent reaction conditions. Both protein preparations were similarly active. Only one viral DNA end was efficiently integrated during Mn(2+)-and Mg(2+)-dependent DNA strand transfer. Water was the predominant nucleophile for Mg(2+)- and Mn(2+)-dependent 3' processing activity. The results underscore functional similarities between EIAV integrase and the previously characterized HIV-1 enzyme.

Chromatography, Affinity↗
PubMed1998

Purification of untagged retroviral integrases by immobilized metal ion affinity chromatography.

We have developed a simple protocol for the purification of untagged retroviral integrases expressed in bacterial cells. The method takes advantage of the inherent ability of the proteins to bind metal ions. The protocol involves an initial enrichment of the protein in the pellet fraction following centrifugation of the lysate after cell lysis. Integrase is then solubilized from the pellet at high salt conditions (1 M) with detergent and applied to a nickel-charged iminodiacetic acid-Sepharose column. The enzyme is eluted from the column with imidazole. The resulting protein, which is 70-80% homogeneous, is subsequently purified to homogeneity on a heparin-Sepharose column. The two-column protocol is easily completed in a day and yields approximately 2 mg of enzymatically active protein per gram of wet cell paste.

Avian Sarcoma Viruses↗
PubMed1995

Analysis of human immunodeficiency virus type 1 integrase mutants.

The human immunodeficiency virus type-1 (HIV-1) integrase protein (IN) mediates the insertion of linear double-stranded viral DNA into the host genome. Mutations in IN can have different effects on the virus life cycle. In this study, Gag-Pol polyprotein processing, Tat synthesis, and viral replication were investigated in integrase-defective HIV-1 mutants. In the absence of IN synthesis, the Gag-Pol polyprotein stability, packaging, and/or processing was reduced. There was limited expression of Tat observed in IN mutants, but no viral replication.

Base Sequence↗
PubMed1996

Catalytic activities of the human T-cell leukemia virus type II integrase.

Despite the widespread nature of HTLV-II in New World populations and intravenous drug users, the enzymatic activities of the pol genes have not been reported. To ascertain the activity of the HTLV-II(G12) integrase (IN), the coding region was isolated and the encoded protein was purified, using nickel-affinity chromatography, to greater than 90% homogeneity. HTLV-II(G12) IN proved active on HTLV-II(G12) and HIV-1 integration and disintegration substrates. Distinct differences in requirements for enzyme concentration for 3'-processing, strand-transfer, and disintegration reactions were observed. Catalysis of integration reactions occurred in the presence of either Mn2+ or Mg2+, although strand-transfer activity preferred Mn2+. In comparison, HTLV-II(G12) IN catalyzed disintegration reactions with almost 10-fold less protein, was not selective for Mn2+ or Mg2+, and tolerated higher NaCl concentrations than integration. HTLV-II(G12) IN was unable to catalyze the "splicing" reaction, which suggests that this may not be an activity ubiquitous to all retroviral integrases.

Animals↗
PubMed1996

Relationships between transposable elements based upon the integrase-transposase domains: is there a common ancestor?

The integrase domain of RNA-mediated elements (class I) and the transposase domain of DNA-mediated transposable elements (class II) were compared. A number of elements contain the DDE signature, which plays an important role in their integration. The possible relationships between mariner-Tc1 and IS elements, retrotransposons, and retroviruses were analyzed from an alignment of this region. The mariner-Tc1 superfamily, and LTR retrotransposons and retroviruses were found to be monophyletic groups. However, the IS elements of bacteria were found in several groups. These results were used to propose an evolutionary history that suggests a common ancestor for some integrases and transposases.

Amino Acid Sequence↗
PubMed2004

Isolation, structure, and HIV-1-integrase inhibitory activity of structurally diverse fungal metabolites.

HIV-1 integrase is a critical enzyme for replication of HIV, and its inhibition is one of the most promising new drug strategies for anti-retroviral therapy, with potentially significant advantages over existing therapies. In this report, a series of HIV-1 inhibitors isolated from the organic extract of fermentations from terrestrial fungi is described. These fungal species, belonging to a variety of genera, were collected from throughout the world following the strict guidelines of Rio Convention on Biodiversity. The polyketide- and terpenoid-derived inhibitors are represented by two naphthoquinones, a biphenyl and two triphenyls, a benzophenone, four aromatics with or without catechol units, a linear aliphatic terpenoid, a diterpenoid, and a sesterterpenoid. These compounds inhibited the coupled and strand-transfer reaction of HIV-1 integrase with an IC(50) value of 0.5-120 micro M. The bioassay-directed isolation, structure elucidation, and HIV-1 inhibitory activity of these compounds are described.

Alkenes↗
PubMed1990

Characterization of an internally initiated integrase protein of HIV-1 produced in E. coli.

In E. coli cells transformed by an expression vector for the production of the protease (PR) integrase (IN) of HIV-1, three vitally encoded proteins were produced: an 11-kDa protein and a 32-kDa protein identified by immunoassays as the mature PR and IN protein, respectively, and an additional protein 15-kDa in size that reacted strongly with an antiserum recognizing a region in the carboxyl half of the IN protein. The kinetics of its synthesis indicated that it was not a degradation product of p32-IN, rather it probably arose from internal initiation at an AUG codon in the middle of the IN gene. Amino terminal sequence analysis of the first 70 residues demonstrated a perfect match with those predicted from the nucleotide sequence, beginning with the methionine codon at position 154 of the integrase gene.

Amino Acid Sequence↗
PubMed1992

The N-terminal region of HIV-1 integrase is required for integration activity, but not for DNA-binding.

HIV-1 integrase binds to both double- and single-stranded DNA with Kd-values of around 20 nM, irrespective of sequence similarities with the termini of the viral LTR. For integration activity, however, the correct LTR sequence of the substrate is required. The putative zinc-binding site present at the N-terminus of the protein is not essential for DNA binding, since deletion mutants of the protein lacking this sequence show similar affinity towards DNA as the wild-type; however, these mutants are not capable of performing the LTR-cleavage and integration reactions. Thus, it appears that the N-terminal part of the integrase is essential for catalytic activity.

Base Sequence↗
PubMed1988

Autonomous DNA binding domains of lambda integrase recognize two different sequence families.

The 40 kd lambda Integrase protein is shown to contain two autonomous DNA binding domains with different sequence specificities. Competition experiments in which the binding activity of Int is assayed through nuclease protection demonstrate the functional independence of the two DNA recognition specificities. Proteolytic cleavage of Int and footprinting analysis of the resulting two major peptides allow the physical separation and identification of two DNA binding domains: an amino-terminal peptide that interacts with "arm-type" sites and a carboxy-terminal peptide that binds to "core-type" sequences. In addition, the data suggest that the two domains can bind DNA simultaneously, consistent with a model in which Integrase would link two disparate DNA sequences.

Amino Acid Sequence↗
PubMed1996

Assaying the activity of HIV-1 integrase with DNA-coated plates.

Integration of reverse transcribed viral DNA of HIV into host chromosomes is mediated by the viral enzyme, integrase. This enzymatic activity can be monitored in vitro by integration of a small labeled DNA (donor) into a second unlabeled DNA (target). The methodology usually involves isotope labeling and gel electrophoresis. To simplify the measurement, a method mimicking enzyme-linked immunosorbent assay (ELISA) procedures was developed. Fragments of DNA were adsorbed directly on 96-well plates and used as the target DNA. The donor was a synthetic 21-bp DNA duplex of HIV-1 U5 LTR; biotin was incorporated into the 5' end of one strand whose two nucleotides at the 3' end were specifically removed during the integration. As a result of integration, the biotin-labeled donor DNA was joined with the target DNA and became immobilized on plates. These integration products were then measured by binding of avidin-alkaline phosphatase on plates. The method is simple and straightforward and can easily be adapted for high throughput screening of integrase inhibitors.

DNA↗
PubMed1991

The oac gene encoding a lipopolysaccharide O-antigen acetylase maps adjacent to the integrase-encoding gene on the genome of Shigella flexneri bacteriophage Sf6.

Lysogens of Shigella flexneri harbouring the temperate bacteriophage, Sf6, have been previously shown to undergo a serotype conversion due to O-acetylation of the O-antigen of the lipopolysaccharide. A partial physical map of the phage genome has been constructed. Analysis of the phage DNA suggests that the phage packages by a headful mechanism and that the mature DNA molecules are terminally redundant. Cloning of the PstI fragments of Sf6 enabled the region encoding the serotype conversion to be localized, showing that this was clearly phage-encoded. The gene was further localized by mutagenesis with Tn5 and the nucleotide sequence of the entire 2693-bp PstI fragment was determined. Two major open reading frames (ORFs) were found capable of encoding proteins of 44.1 and 37.2 kDa. The latter corresponds to the O-antigen acetylase and its gene has been designated oac. The oac gene is capable of converting Sh. flexneri serotypes X, Y, 1a and 4a to 3a, 3b, 1b and 4b, respectively. The Oac protein bears a high degree of homology to the NodX protein of Rhizobium leguminosarum suggesting that it, too, may be a sugar acetylase. The second ORF immediately upstream from oac corresponds to the bacteriophage Sf6 integrase responsible for chromosomal integration and is highly homologous to the integrases of Escherichia coli bacteriophages P4 and phi 80, but less closely related to those of P1, P2, P22, 186 and lambda.

Acetylesterase↗
PubMed2005

Molecular dynamics studies of the full-length integrase-DNA complex.

We have carried out a molecular dynamics (MD) simulation of full-length HIV-1 integrase (IN) dimer complexed with viral DNA with the aim of gaining information about the enzyme motion and investigating the movement of the catalytic flexible loop (residues 140-149) thought to be essential in the catalytic mechanism of IN. During the simulation, we observed quite a different behavior of this region in the presence or absence of the viral DNA. In particular, the MD results underline the crucial role of the residue Tyr143 in the mechanism of integration of viral DNA into the host chromosome. The present findings confirm the experimental data (e.g., site-directed mutagenesis experiments) showing that the loop is involved in the integration reactions and its mobility is correlated with the catalytic activity of HIV-1 integrase.

Catalytic Domain↗
PubMed2004

Synthesis and HIV-1 integrase inhibitory activity of dimeric and tetrameric analogs of indolicidin.

We found that indolicidin, a natural antimicrobial peptide, has HIV-1 integrase inhibitory activity. Subsequently, we also discovered analogs of indolicidin with substantially higher inhibitory potency. The dimers and tetramers of the most active sequence (ILPWKWPWWPWPP) were prepared by connection of the monomers' C-terminal ends, using lysine as a linker. The inhibitory potency of the dimeric peptide is higher than the monomeric peptide. The tetrameric peptide, prepared by connection of two dimers at C-ends using again lysine as the linker, is the most potent integrase inhibitor with IC(50) value of 0.6 microM for both 3'-end processing and strand transfer.

Anti-HIV Agents↗
PubMed2005

A series of 5-(5,6)-dihydrouracil substituted 8-hydroxy-[1,6]naphthyridine-7-carboxylic acid 4-fluorobenzylamide inhibitors of HIV-1 integrase and viral replication in cells.

Introduction of a 5,6-dihydrouracil functionality in the 5-position of N-(4-fluorobenzyl)-8-hydroxy-[1,6]naphthyridine-7-carboxamide 1 led to a series of highly active HIV-1 integrase inhibitors. These compounds displayed low nanomolar activity in inhibiting both the strand transfer process of HIV-1 integrase and viral replication in cells. Compound 11 is a 150-fold more potent antiviral agent than 1, with a CIC(95) of 40 nM in the presence of human serum. It displays good pharmacokinetics when dosed in rats and dogs.

Animals↗
PubMed2006

Design, synthesis, and SAR studies of novel and highly active tri-cyclic HIV integrase inhibitors.

A novel class of tri-cyclic HIV integrase inhibitors were designed based on conformational analysis of 1,6-naphthyridine carboxamide compound L-870810 and docking the designed inhibitor into the active site of our integrase enzyme model. The efficient syntheses of pyrroloquinoline tri-cyclic analogs are described. The SAR studies resulted in the identification of a lead compound that is more potent and more soluble than L-870810.

Drug Design↗
PubMed2005

The C-terminal chromodomain-like module in the integrase domain is crucial for high transposition efficiency of the retrotransposon MAGGY.

MAGGY is a Ty3/Gypsy retrotransposon, which was identified in the rice blast fungus Magnaporthe oryzae. Some Ty3/Gypsy retrotransposons, including MAGGY, contain a chromodomain-like module (CLM) in the C-terminus of the integrase domain. We have made a series of MAGGY mutants to examine the role of the CLM in the transposition activity of the element. Introduction of a mutation at different positions in the MAGGY integrase revealed that a loss or alteration of the CLM resulted in a drastic decrease in the transposition activity of the element. Our results indicate that the CLM may confer high transposition activity to the element.

Amino Acid Sequence↗