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At least 343 records · Page 19Linked to original sources
PubMed2006

Identification of pseudo attP sites for phage phiC31 integrase in bovine genome.

Streptomyces phage phiC31 integrase was found to mediate site-specific integration of foreign genes at pseudo attP sites of genomes in human, mouse, rat, and Drosophila. This paper reports that phiC31 integrase can also mediate homologous recombination between attB and pseudo attP sites in bovine cells and foreign gene integration was increased at least 2-fold in bovine fibroblasts or Madin-Darby bovine kidney (MDBK) cells. Two intrinsic pseudo attP sites named BpsF1 and BpsM1 located in the inter-gene regions on chromosome 28 and 19, respectively, were identified in bovine genome. These pseudo attP sites shared similar characteristics with those from other species as previously described. Our study demonstrated that the phiC31 integrase system provides a new potential for genetic engineering of the bovine genome and might be beneficial for the research on ruminant.

Animals↗
PubMed2006

Beta-diketo acids with purine nucleobase scaffolds: novel, selective inhibitors of the strand transfer step of HIV integrase.

The HIV pol gene encodes three viral enzymes that are required for its replication. While drug discovery involving the viral targets, reverse transcriptase and protease, has resulted in useful therapeutic agents, such efforts on HIV integrase have not produced a single FDA-approved drug. In the work focused on the discovery of inhibitors of HIV integrase, we have synthesized new beta-diketo acids with purine nucleobase scaffolds that are potent inhibitors of the strand transfer steps of wild-type HIV-1 integrase.

Acids↗
PubMed2006

Exploration of the diketoacid integrase inhibitor chemotype leading to the discovery of the anilide-ketoacids chemotype.

Integrase is one of three enzymes expressed by HIV and represents a validated target for therapy. A previous study of the diketoacid-based chemotype suggested that there are two aryl-binding domains on integrase. In this study, modifications to the indole-based diketoacid chemotype are explored. It is demonstrated that the indole group can be replaced with secondary but not tertiary (e.g., N-methyl) aniline-based amides without sacrificing in vitro inhibitory activity. The difference in activity between the secondary and tertiary amides is most likely due to the opposite conformational preferences of the amide bonds, s-trans for the secondary-amide and s-cis for the tertiary-amide. However, it was found that the conformational preference of the tertiary amide can be reversed by incorporating the amide nitrogen atom into an indoline heterocycle, resulting in very potent integrase inhibitors.

Amides↗
PubMed2004

Dimerization of bacteriophage P2 integrase is not required for binding to its DNA target but for its biological activity.

Coliphage P2 integrates into the host chromosome upon lysogenization via site-specific recombination mediated by the phage integrase (Int). P2 integrase belongs to the tyrosine family of recombinases. In this work, it is shown that P2 integrase forms dimers but not oligomers in the absence of its DNA target. Furthermore, the C-terminal end of the protein and amino acid (aa) E197 have been found to be involved in dimerization. Amino acid E197 is located in a conserved region of the tyrosine recombinases that has not previously been implicated in dimerization. The dimerization deficient mutants were unaffected in binding to its phage attachment site (attP) substrate, but had a reduced ability to complement an int-defective prophage.

Amino Acid Sequence↗
PubMed2006

Identification and characterization of the chromatin-binding domains of the HIV-1 integrase interactor LEDGF/p75.

Depletion of the transcriptional co-activator LEDGF/p75 by RNA interference alters the genome-wide pattern of HIV-1 integration, reducing integration into active genes, reducing integration into LEDGF/p75-regulated genes, and increasing integration into G+C-rich sequences. LEDGF/p75 is also able to act as a molecular tether linking HIV-1 integrase protein to chromatin, a phenomenon likely to underlie the integration site distribution effects. The LEDGF/p75 integrase-binding domain has been established but the domain or domains responsible for the chromatin-binding component of tethering are unknown. Here, we identify and characterize these domains. Complementary methods were used to assess condensed and uncondensed chromatin, and to determine the stringency of chromatin binding. Immuno-localization analyses revealed that an N-terminal PWWP domain and its beta-barrel substructure are needed for binding to metaphase chromatin. However, the PWWP domain is insufficient to transfer metaphase chromatin binding to green fluorescent protein, which requires addition of a downstream charged region (CR1). Biochemical analysis showed that full-length LEDGF/p75 resists Triton X-100 extraction from chromatin. To transfer Triton-resistant chromatin binding to green fluorescent protein, PWWP-CR1 is necessary but not sufficient. Further inclusion of a tandem pair of AT-hooks in combination with at least one of two identified downstream charged regions (CR2 or CR3) is needed. Deletion of just the PWWP or the AT-hook domain from full-length LEDGF/p75 reduced Triton-resistant chromatin binding, while deletion of both elements abolished it, underscoring their dominant and cooperative role. The results establish a molecular mechanism for LEDGF/p75-mediated tethering of HIV-1 integrase to chromatin.

Amino Acids↗
PubMed2006

Identification of the LEDGF/p75 binding site in HIV-1 integrase.

Lens epithelium-derived growth factor (LEDGF)/p75 is an important cellular co-factor for human immunodeficiency virus (HIV) replication. We originally identified LEDGF/p75 as a binding partner of integrase (IN) in human cells. The interaction has been mapped to the integrase-binding domain (IBD) of LEDGF/p75 located in the C-terminal part. We have subsequently shown that IN carrying the Q168A mutation remains enzymatically active but is impaired for interaction with LEDGF/p75. To map the integrase/LEDGF interface in more detail, we have now identified and characterized two regions within the enzyme involved in the interaction with LEDGF/p75. The first region centers around residues W131 and W132 while the second extends from I161 up to E170. For the different IN mutants the interaction with LEDGF/p75 and the enzymatic activities were determined. IN(W131A), IN(I161A), IN(R166A), IN(Q168A) and IN(E170A) are impaired for interaction with LEDGF/p75, but retain 3' processing and strand transfer activities. Due to impaired integration, an HIV-1 strain containing the W131A mutation in IN displays reduced replication capacity, whereas virus carrying IN(Q168A) is replication defective. Comparison of the wild-type IN-LEDGF/p75 co-crystal structure with that of the modelled structure of the IN(Q168A) and IN(W131A) mutant integrases corroborated our experimental data.

Amino Acid Sequence↗
PubMed2004

Globoidnan A: a lignan from Eucalyptus globoidea inhibits HIV integrase.

An HTS campaign aimed at the identification of inhibitors of HIV integrase showed that the methanol extract from the buds of a Eucalyptus globoidea was active. Bioassay guided fractionation of this extract resulted in the purification and structural elucidation of the lignan, globoidnan A (1) as the only compound in the extract responsible for the inhibition of HIV integrase. The compound was found to inhibit the combined 3' processing and strand transfer activity of HIV integrase with an IC50=0.64 microM.

Eucalyptus↗
PubMed2005

A eukaryotic gene family related to retroelement integrases.

Proteins encoded by mobile genetic elements occasionally assume cellular roles. Telomerase, for example, is a reverse transcriptase that replicates chromosome ends, and Rag1 is a transposase that mediates immunoglobulin gene rearrangements. In this article, we report cellular genes related to integrases that are not associated with a retrovirus or retrotransposon. These integrases are found in diverse eukaryotes and are evolving under functional constraint. We propose that the cellular integrases have assumed a host role and, like their retroelement counterparts, probably function in DNA metabolism.

Amino Acid Sequence↗
PubMed1996

The catalytic domain of human immunodeficiency virus integrase: ordered active site in the F185H mutant.

We solved the structure and traced the complete active site of the catalytic domain of the human immunodeficiency virus type 1 integrase (HIV-1 IN) with the F185H mutation. The only previously available crystal structure, the F185K mutant of this domain, lacks one of the catalytically important residues, E152, located in a stretch of 12 disordered residues [Dyda et al. (1994) Science 266, 1981-1986]. It is clear, however, that the active site of HIV-1 IN observed in either structure cannot correspond to that of the functional enzyme, since the cluster of three conserved carboxylic acids does not create a proper metal-binding site. The conformation of the loop was compared with two different conformations found in the catalytic domain of the related avian sarcoma virus integrase [Bujacz et al. (1995) J. Mol. Biol. 253, 333-346]. Flexibility of the active site region of integrases may be required in order for the enzyme to assume a functional conformation in the presence of substrate and/or cofactors.

Binding Sites↗
PubMed1999

HIV integrase structure and function.

HIV integrase consists of three domains, the structures of which have been individually determined by X-ray crystallography or NMR spectroscopy. The core domain, spanning residues 50-212, is responsible for the catalytic activity of the enzyme. The crystal structure of a dimer of this domain shows similarity to other proteins that carry out polynucleotidyl transfer, including MuA transposase and RNase H. The small N-terminal domain folds into a dimeric helix-turn-helix structure, which is stabilized by the coordination of zinc with conserved His and Cys residues. The function of this domain is unclear; however, it is required for integration activity and enhances tetramerization in the context of the full-length integrase. The C-terminal domain, which has an SH3-like fold, is involved in DNA binding. The structure of this domain reveals a large saddle-shaped cleft that is formed by dimerization. This cleft contains a number of positively charged residues, and its dimensions are appropriate for accommodating a double-stranded DNA helix. Although the C-terminal domain was originally believed to be involved in target DNA binding, more recent evidence suggests that it may bind to both the ends of the viral DNA and to the target DNA. Although the individual domain structures provide some insights into the function of the protein, a more detailed understanding of the complete mechanism by which integrase binds, cleaves, and transfers DNA requires a greater knowledge of how these domains are arranged in the active multimer.

Animals↗
PubMed2000

Insertion of bacteriophage phiFSW into the chromosome of Lactobacillus casei strain Shirota (S-1): characterization of the attachment sites and the integrase gene.

The integrase gene (int) on the genome of φFSW, which is a temperate bacteriophage of Lactobacillus casei strain Shirota (formerly denoted as S-1), and the four attachment sites on the genomes of the phage and its host were characterized by sequencing. The φFSW integrase was found to belong to the integrase family of site-specific tyrosine recombinase. The attachment sites shared a 40bp common core within which an integrative site-specific recombination occurs. The common core was flanked on one side by an additional segment of high sequence similarity. An integration plasmid, consisting of int, the phage attachment site (attP), and a selectable marker, inserted stably into the bacterial attachment site (attB) within the common core, as did the complete prophage genome at a frequency of more than 10(3)/microg of plasmid DNA. This plasmid was used as a test system for a preliminary mutational analysis of int and attP. The attB common core was located within and near the end of an open reading frame that appears to encode a homolog to glucose 6-phosphate isomerase, an enzyme of the glycolytic pathway. It is unlikely that the prophage integration inactivates this protein, since a change of only the C-terminal amino acid is predicted because of the sequence similarity between attP and attB.

Amino Acid Sequence↗
PubMed1996

Retroviral integrases and their cousins.

The recently determined structures of the catalytic domains of HIV integrase, avian sarcoma virus integrase and the Mu transposase are strikingly similar to each other and also exhibit significant similarity to several nucleases. All these enzymes of cut polynucleotides, leaving 3'OH and 5'PO4 groups. The integrase and transposase also possess a strand-transfer activity that splices DNA. The structural similarities among members of this superfamily of polynucleotidyl transferases suggest that they share a similar mechanism of catalysis.

Bacterial Proteins↗
PubMed2000

Chemical and enzymatic modifications of integric acid and HIV-1 integrase inhibitory activity.

Integric acid (1), an acyl eremophilane sesquiterpenoid, was identified as an inhibitor of HIV-1 integrase, the enzyme responsible for provirus entry into the host cell nucleus and integration in to the host genome. Chemical and enzymatic modification of integric acid led to the preparation of several selective chemical derivatives of integric acid. Preparation, HIV-1 inhibitory activity, and the structure-activity relationship against coupled and strand transfer assays are described. It appears that most of the groups present in the natural product are required for inhibition of HIV-1 integrase strand transfer activity. In contrast, inhibition of 3' processing activity is less stringent suggesting distinct SAR for the two integrase reactions.

Amides↗
PubMed2002

New class of HIV integrase inhibitors that block viral replication in cell culture.

BACKGROUND: To improve the existing combination therapies of infection with the human immunodeficiency virus (HIV) and to cope with virus strains that are resistant to multiple drugs, we initiated a search for effective inhibitors of HIV integrase, the enzyme responsible for inserting the viral cDNA into the host cell chromosome. RESULTS: We have now identified a series of 5H-pyrano[2,3-d:-6,5-d']dipyrimidines that block the replication of various strains of HIV-1 and HIV-2. The most potent congener, 5-(4-nitrophenyl)-2,8-dithiol-4,6-dihydroxy-5H-pyrano[2,3-d:-6,5-d']dipyrimidine (V-165), inhibited the replication of HIV-1(III(B)) in MT-4 cells at a 50% effective concentration (EC(50)) of 8.9 microM, which is 14-fold below its cytotoxic concentration. V-165 was equally active against virus strains that were resistant toward inhibitors of viral entry or reverse transcriptase. In combination regimens in cell culture, V-165 acted subsynergistically with zidovudine or nelfinavir and synergistically with nevirapine. V-165 inhibited both reverse transcriptase and integrase activities in enzymatic assays at micromolar concentrations, but only a close correlation was found between the anti-HIV activity observed in cell culture and the inhibitory activity in the integrase strand transfer assays. Time-of-addition experiments indicated that V-165 interfered with the viral replication cycle at a time point coinciding with integration. Quantitative Alu-PCR corroborated that the anti-HIV activity of V-165 is based upon the inhibition of proviral DNA integration. CONCLUSIONS: Based on their mode of action, which is different from that of clinically approved anti-HIV drugs, PDPs are good candidates for further development into new drugs and to be included in future combination regimens.

Cell Line↗
PubMed1996

The catalytic domain of avian sarcoma virus integrase: conformation of the active-site residues in the presence of divalent cations.

BACKGROUND: Members of the structurally-related superfamily of enzymes that includes RNase H, RuvC resolvase, MuA transposase, and retroviral integrase require divalent cations for enzymatic activity. So far, cation positions are reported in the X-ray crystal structures of only two of these proteins, E. coli and human immunodeficiency virus 1 (HIV-1) RNase H. Details of the placement of metal ions in the active site of retroviral integrases are necessary for the understanding of the catalytic mechanism of these enzymes. RESULTS: The structure of the enzymatically active catalytic domain (residues 52-207) of avian sarcoma virus integrase (ASV IN) has been solved in the presence of divalent cations (Mn2+ or Mg2+), at 1.7-2.2 A resolution. A single ion of either type interacts with the carboxylate groups of the active site aspartates and uses four water molecules to complete its octahedral coordination. The placement of the aspartate side chains and metal ions is very similar to that observed in the RNase H members of this superfamily; however, the conformation of the catalytic aspartates in the active site of ASV IN differs significantly from that reported for the analogous residues in HIV-1 IN. CONCLUSIONS: Binding of the required metal ions does not lead to significant structural modifications in the active site of the catalytic domain of ASV IN. This indicates that at least one metal-binding site is preformed in the structure, and suggests that the observed constellation of the acidic residues represents a catalytically competent active site. Only a single divalent cation was observed even at extremely high concentrations of the metals. We conclude that either only one metal ion is needed for catalysis, or that a second metal-binding site can only exist in the presence of substrate and/or other domains of the protein. The unexpected differences between the active sites of ASV IN and HIV-1 IN remain unexplained; they may reflect the effects of crystal contacts on the active site of HIV-1 IN, or a tendency for structural polymorphism.

Avian Sarcoma Viruses↗
PubMed2000

Determinants of Mg2+-dependent activities of recombinant human immunodeficiency virus type 1 integrase.

The relationship between Mg(2+)-dependent activity and the self-assembly state of HIV-1 integrase was investigated using different protein preparations. The first preparations, IN(CHAPS) and IN(dial), were purified in the presence of detergent, but in the case of IN(dial), the detergent was removed during a final dialysis. The third preparation, IN(zn), was purified without any detergent. The three preparations displayed comparable Mn(2+)-dependent activities. In contrast, the Mg(2+)-dependent activity that reflects a more realistic view of the physiological activity strongly depended on the preparation. IN(CHAPS) was not capable of using Mg(2+) as a cofactor, whereas IN(zn) was highly active under the same conditions. In the accompanying paper [Deprez, E., et al. (2000) Biochemistry 39, 9275-9284], we used time-resolved fluorescence anisotropy to demonstrate that IN(CHAPS) was monomeric at the concentration of enzymatic assays. Here, we show that IN(zn) was homogeneously tetrameric under similar conditions. Moreover, IN(dial) that exhibited an intermediary Mg(2+)-dependent activity existed in a monomer-multimer equilibrium. The level of Mg(2+)- but not Mn(2+)-dependent activity of IN(dial) was altered by addition of detergent which plays a detrimental role in the maintenance of the oligomeric organization. Our results indicate that the ability of integrase to use Mg(2+) as a cofactor is related to its self-assembly state in solution, whereas Mn(2+)-dependent activity is not. Finally, the oligomeric IN(zn) was capable of binding efficiently to DNA regardless of the cationic cofactor, whereas the monomeric IN(CHAPS) strictly required Mn(2+). Thus, we propose that a specific conformation of integrase is a prerequisite for its binding to DNA in the presence of Mg(2+).

Cholic Acids↗
PubMed2003

Energetics of sequence-specific protein-DNA association: conformational stability of the DNA binding domain of integrase Tn916 and its cognate DNA duplex.

Sequence-specific DNA recognition by bacterial integrase Tn916 involves structural rearrangements of both the protein and the DNA duplex. Energetic contributions from changes of conformation, thermal motions and soft vibrational modi of the protein, the DNA, and the complex significantly influence the energetic profile of protein-DNA association. Understanding the energetics of such a complicated system requires not only a detailed calorimetric investigation of the association reaction but also of the components in isolation. Here we report on the conformational stability of the integrase Tn916 DNA binding domain and its cognate 13 base pair target DNA duplex. Using a combination of temperature and denaturant induced unfolding experiments, we find that the 74-residue DNA binding domain is compact and unfolds cooperatively with only small deviation from two-state behavior. Scanning calorimetry reveals an increase of the heat capacity of the native protein attributable to increased thermal fluctuations. From the combined calorimetric and spectroscopic experiments, the parameters of protein unfolding are T(m) = 43.8 +/- 0.3 degrees C, DeltaH(m) = 255 +/- 18 kJ mol(-1), DeltaS(m) = 0.80 +/- 0.06 kJ mol(-1), and DeltaC(p) = 5.0 +/- 0.8 kJ K(-1) mol(-1). The DNA target duplex displays a thermodynamic signature typical of short oligonucleotide duplexes: significant heat absorption due to end fraying and twisting precedes cooperative unfolding and dissociation. The parameters for DNA unfolding and dissociation are DeltaH(m) = 335 +/- 4 kJ mol(-1) and DeltaC(p) = 2.7 +/- 0.9 kJ K(-(1) mol(-1). The results reported here have been instrumental in interpreting the thermodynamic features of the association reaction of the integrase with its 13 base pair target DNA duplex reported in the accompanying paper [Milev et al. (2003) Biochemistry 42, 3481-3491].

Base Sequence↗
PubMed2003

Dynamic, thermodynamic, and kinetic basis for recognition and transformation of DNA by human immunodeficiency virus type 1 integrase.

Specific interactions between retroviral integrase (IN) and long terminal repeats are required for insertion of viral DNA into the host genome. To characterize quantitatively the determinants of substrate specificity, we used a method based on a stepwise increase in ligand complexity. This allowed an estimation of the relative contributions of each nucleotide from oligonucleotides to the total affinity for IN. The interaction of HIV-1 integrase with specific (containing sequences from the LTR) or nonspecific oligonucleotides was analyzed using a thermodynamic model. Integrase interacted with oligonucleotides through a superposition of weak contacts with their bases, and more importantly, with the internucleotide phosphate groups. All these structural components contributed in a combined way to the free energy of binding with the major contribution made by the conserved 3'-terminal GT, and after its removal, by the CA dinucleotide. In contrast to nonspecific oligonucleotides that inhibited the reaction catalyzed by IN, specific oligonucleotides enhanced the activity, probably owing to the effect of sequence-specific ligands on the dynamic equilibrium between the oligomeric forms of IN. However, after preactivation of IN by incubation with Mn(2+), the specific oligonucleotides were also able to inhibit the processing reaction. We found that nonspecific interactions of IN with DNA provide approximately 8 orders of magnitude in the affinity (Delta G degrees approximately equal to -10.3 kcal/mol), while the relative contribution of specific nucleotides of the substrate corresponds to approximately 1.5 orders of magnitude (Delta G degrees approximately equal to - 2.0 kcal/mol). Formation of the Michaelis complex between IN and specific DNA cannot by itself account for the major contribution of enzyme specificity, which lies in the k(cat) term; the rate is increased by more than 5 orders of magnitude upon transition from nonspecific to specific oligonucleotides.

DNA, Single-Stranded↗