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At least 19 recordsLinked to original sources
PubMed1986

Mutational analysis of integrase arm-type binding sites of bacteriophage lambda. Integration and excision involve distinct interactions of integrase with arm-type sites.

Integrative recombination between specific attachment (att) regions of the bacteriophage lambda genome (attP) and the Escherichia coli genome (attB) results in a prophage flanked by the hybrid recombinant sites attL and attR. Each att site contains sequences to which proteins involved in recombination bind. Using site-directed mutagenesis, we have constructed a related set of point mutations within each of the five Int "arm-type" binding sites located within attP, attL and attR. Footprint analyses of binding demonstrate that mutating the arm-type sites significantly disrupts the binding of Int. Recombination analyses of mutant att sites in vivo and in vitro demonstrate that only three wild-type arm-type sites within attP are required for efficient integrative recombination. Similar analyses demonstrate that efficient excision can occur with two other different sets of wild-type arm-type sites in attL and attR. These results demonstrate that integrative and excisive recombination may involve interactions of Int with distinct and different subsets of arm-type sites.

Attachment Sites, Microbiological↗
PubMed1992

Mapping of immunodominant epitopes of the HIV-1 and HIV-2 integrase proteins by recombinant proteins and synthetic peptides.

Different parts of the human immunodeficiency virus type 1 and type 2 (HIV-1 and HIV-2) integrase proteins were expressed as TrpE fusion proteins in Escherichia coli and used to screen human sera. In the immunoblot, all HIV/integrase-positive human sera tested reacted with the carboxy-terminal third of the integrase protein. Furthermore, they crossreacted with the same part of the heterologous protein. Half (50%) of the HIV-1/integrase-positive sera additionally detected antigenic epitopes in the amino-terminal third of the HIV-1 protein. Two of the recombinant proteins were used to generate polyclonal rabbit sera, which react with type-common epitopes of both integrase proteins. To map the B-cell epitopes of the HIV integrase proteins in more detail, overlapping decapeptides representing the entire integrase proteins of HIV-1 and HIV-2 were synthesized and used in a pin-based oligopeptide ELISA to scan human sera. This method can define three potential immunogenic epitopes of the HIV-1 integrase and one potential epitope of the HIV-2 integrase. The immunodominant epitopes of the HIV-1 integrase, one localized in the amino-terminal (IDKAQDEHEKYHSNWRAM), one in the central (QMAVFIHNFKRKGGIGGY), and one in the carboxy-terminal (AVVIQDNSDIKVVPRRK) part of the protein were synthesized as oligopeptides and used to test a larger panel of human sera in ELISA (156 HIV-1+ sera and 104 HIV-1- sera). The amino- and the carboxy-terminal epitopes were of equivalent reactivity, while the central part of the HIV-1 integrase seems to be less immunogenic. Nearly 90% of the HIV-1/integrase-positive human sera could be detected by a combination of these three peptides.

Amino Acid Sequence↗
PubMed1990

Expression of HIV-1 integrase in E. coli: immunological analysis of the recombinant protein.

Sequences encoding the human immunodeficiency virus type 1 (HIV-1) integrase gene have been cloned and expressed in Escherichia coli. The expressed protein is a lambda cII fusion protein of 37 kD containing the carboxyl-terminal 23 [corrected] amino acids of reverse transcriptase fused to the entire integrase sequence and is insoluble, a feature which allows partial purification away from soluble bacterial proteins. As judged by its reactivity with HIV positive sera in Western blot and in enzyme-linked immunosorbent assay (ELISA), the recombinant integrase retains antigenicity similar to native protein. Additionally, ELISA data obtained with the cloned protein indicate that patients infected with HIV-1 who are at different stages of progression to AIDS have antibodies reactive with the cloned integrase. HIV-2 positive human sera are also reactive with the cloned integrase. Rabbit antibodies produced against the recombinant protein react both by ELISA and Western blot with the homologous bacterially expressed protein, recognize both virion HIV-1 integrase and reverse transcriptase in Western blots, and immunoprecipitate an HIV-1 virion protein of 34 kD. Unlike human antisera from patients infected with HIV-1 or HIV-2 which are frequently reactive with both HIV-1 and HIV-2 integrase, the rabbit antibodies are type specific, reacting with HIV-1, but not with HIV-2 integrase by Western blot.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Requirement of active human immunodeficiency virus type 1 integrase enzyme for productive infection of human T-lymphoid cells.

The human immunodeficiency virus type 1 (HIV-1) integrase enzyme exhibits significant amino acid sequence conservation with integrase proteins of other retroviruses. We introduced specific amino acid substitutions at a number of the conserved residue positions of recombinant HIV-1 integrase. Some of these substitutions resulted in proteins which were not able to be purified in the same manner as the wild-type enzyme, and these were not studied further. The remaining mutant enzymes were assessed for their abilities to perform functions characteristic of the integrase protein. These included specific removal of the terminal dinucleotides from oligonucleotide substrates representative of the viral U5-long terminal repeat, nonspecific cleavage of oligonucleotide substrates, and mediation of the strand transfer (integration) reaction. Substitution at position 43, within the protein's zinc finger motif region, resulted in an enzyme with reduced specificity for cleavage of the terminal dinucleotide. In addition, a double substitution of aspartic acid and glutamine for valine and glutamic acid, respectively, at positions 151 and 152 within the D,D(35)E motif region rendered the integrase protein inactive for all of its functions. The introduction of this double substitution into an infectious HIV-1 provirus yielded a mutant virus that was incapable of productively infecting human T-lymphoid cells in culture.

Amino Acid Sequence↗
PubMed1988

Suicide recombination substrates yield covalent lambda integrase-DNA complexes and lead to identification of the active site tyrosine.

High levels of covalent integrase-DNA complexes accumulate when suicide substrates containing a medial nick within the overlap region are nicked by lambda integrase protein. The tyrosine residue at position 342 is shown to form a covalent bond with DNA at the sites of strand exchange. A mutant integrase in which this tyrosine is changed to phenylalanine is devoid of both topoisomerase and recombinase activity but still binds to both core- and arm-type DNA binding sites with an affinity comparable to wild-type integrase. Tyrosine-342 is located within a 40-amino acid region that is conserved among 15 known recombinases comprising the "integrase family." The present results show that this small region of homology participates in catalysis of strand transfer.

Amino Acid Sequence↗
PubMed1992

A frog virus 3 gene codes for a protein containing the motif characteristic of the INT family of integrases.

The integrase (INT) family of bacteriophage coded integrase-recombinase proteins are responsible for catalyzing strand exchange between DNA molecules and play an important role in the DNA replication of many bacteriophages. Within the frog virus 3 (FV3) genome we have identified an open reading frame (ORF) of which the deduced amino acid sequence contains a motif characteristic of the INT family of integrases-recombinases. The ORF consists of 825 bp which codes for a protein of 275 amino acids with a predicted Mr of 29,945. RNA transcribed from this ORF during virus infection was detected by Northern blot analysis and it is a delayed early message of approximately 1100 bases. The 5' and 3' ends of the putative FV3 integrase-recombinase transcript were mapped. The transcriptional start site is preceded by a presumptive TATA box, and a region of hyphenated dyad symmetry is present at the 3' end of the message. A protein with an Mr of approximately 30,500 was synthesized by a rabbit reticulocyte lysate programmed with capped runoff transcripts from the cloned gene, indicating that the ORF can be transcribed into a message coding for a viral protein. In the FV3 life cycle, DNA replication occurs in a large complex formed through the recombination of small viral DNA molecules. Thus, at this stage, DNA replication and recombination are interlinked. Resolution of concatameric DNA is required for the packaging of genomes into virus particles. The putative FV3 INT gene may be involved in one or more of these functions.

Amino Acid Sequence↗
PubMed1991

Prevention of human immunodeficiency virus type 1 integrase expression in Escherichia coli by a ribozyme.

Ribozymes are potentially very powerful agents for perturbing intracellular gene expression. However, pilot experiments in eukaryotes have met with mixed success. We now report that a ribozyme designed to cleave the integrase gene of the human immunodeficiency virus (HIV), when transcribed from a plasmid in Escherichia coli, led to destruction of integrase RNA and complete blockage of integrase protein synthesis. These results indicate that ribozymes can be used to study intracellular gene expression in bacteria and that the HIV-1 integrase gene may be a useful target for therapeutic ribozymes.

Base Sequence↗
PubMed1992

Reversal of integration and DNA splicing mediated by integrase of human immunodeficiency virus.

In retroviral integration, the viral integration protein (integrase) mediates a concerted DNA cleavage-ligation reaction in which the target DNA is cleaved and the resulting 5' ends of target DNA are joined to the 3' ends of viral DNA. Through an oligonucleotide substrate that mimics the recombination intermediate formed by this initial cleavage-ligation reaction, the purified integrase of human immunodeficiency virus was shown to promote the same reaction in reverse, a process called disintegration. Analysis of a set of structurally related substrates showed that integrase could promote a range of DNA cleavage-ligation reactions. When the viral DNA component of the disintegration substrate was single-stranded, integrase could mediate a DNA splicing reaction analogous to RNA splicing.

Base Sequence↗
PubMed1992

Site-specific deletion and rearrangement of integron insert genes catalyzed by the integron DNA integrase.

Deletion of individual antibiotic resistance genes found within the variable region of integrons is demonstrated. Evidence for gene duplications and rearrangements resulting from the insertion of gene units at new locations is also presented. Deletion, duplication, and rearrangement occur only in the presence of the integron-encoded DNA integrase. These events are precise and involve loss or gain of one or more complete insert units or gene cassettes. This confirms the recent definition of gene cassettes as consisting of the gene coding sequences, all except the last 7 bases of the 59-base element found at the 3' end of the gene, and the core site located 5' to the gene (Hall et al., Mol. Microbiol. 5:1941-1959, 1991) and demonstrates that individual gene cassettes are functional units which can be independently mobilized. Both deletions and duplications can be generated by integrase-mediated cointegrate formation followed by integrase-mediated resolution involving a different pair of sites. However, deletion occurs 10 times more frequently than duplication, and we propose that the majority of deletion events are likely to involve integrase-dependent excision of the gene unit to generate a circular gene cassette. The implications of these findings in understanding the evolution of integrons and the spread of antibiotic resistance genes in bacterial populations is discussed.

Base Sequence↗
PubMed1976

Significance of constitutive integrase synthesis.

One conceivable function for constitutive integrase formation by lambda prophage is to stabilize the inserted state by catalyzing reinsertion of prophages that are accidentally excised. As this hypothesis implies a dynamic equilibrium betwen inserted and noninserted DNA, the existence of such an equilibrium is explored. By examining the frequency with which prophages appear in an initially unoccupied chromosomal site of a lysogenic bacterium in which the prophage attachment site is duplicated, the off-rate is estimated as less than 10(-2) per generation for wild-type lambda, and less than 4 x 10(-4) for N- mutants of lambda. From the rate of integrase-catalyzed haploidization of certain partial diploid strains, the rate of spontaneous integrase activity is estimated as 3 x 10(-3) per generation. From these values I conclude that constitutive integrase will not appreciably stabilize the inserted state by virtue of its known activity.

Coliphages↗
PubMed1992

Zinc binding by retroviral integrase.

Zinc binding by integrase from Moloney murine leukaemia virus and a protein A fusion protein containing integrase from human immunodeficiency virus type 1 was demonstrated by a zinc blotting technique using 65ZnCl2. Autoradiography revealed a clear band that was absent from the appropriate controls. This band co-migrated with the major band in Coomassie-stained gels and in immunoblots. This binding activity was retained in the presence of competing divalent cations and was sensitive to oxidation. This is the first demonstration of zinc binding by intact retroviral integrase.

Amino Acid Sequence↗
PubMedDate not supplied

A protein binding to the J kappa recombination sequence of immunoglobulin genes contains a sequence related to the integrase motif.

Site-specific recombination requires conserved DNA sequences specific to each system, and system-specific proteins that recognize specific DNA sequences. The site-specific recombinases seem to fall into at least two families, based on their protein structure and chemistry of strand breakage. One of these is the resolvase-invertase family, members of which seem to form a serine-phosphate linkage with DNA. Members of the other family, called the integrase family, contain a conserved tyrosine residue that forms a covalent linkage with the 3'-phosphate of DNA at the site of recombination. Structural comparison of integrases shows that these proteins share a highly conserved 40-residue motif. V-(D)-J recombination of the immunoglobulin gene requires conserved recombination signal sequences (RS) of a heptamer CACTGTG and a T-rich nonamer GGTTTTTGT, which are separated by a spacer sequence of either 12 or 23 bases We have recently purified, almost to homogeneity, a protein that specifically binds to the immunoglobulin J kappa RS containing the 23-base-pair spacer sequence. By synthesizing probes on the basis of partial amino-acid sequences of the purified protein, we have now isolated and characterized the complementary DNA of this protein. The amino-acid sequence deduced from the cDNA sequence reveals that the J kappa RS-binding protein has a sequence similar to the 40-residue motif of integrases of phages, bacteria and yeast, indicating that this protein could be involved in V-(D)-J recombination as a recombinase.

Amino Acid Sequence↗
PubMed1992

Evidence for a second conserved arginine residue in the integrase family of recombination proteins.

This study was designed to search for new regions of similarity in the integrase family of recombination proteins which consists of 28 members found in bacteria and yeast. A computer method based on an information content analysis has been used to align local regions of homology in the set of unaligned protein sequences from this family. Among the aligned regions with high information content were those containing the known conserved histidine, arginine and tyrosine residues. In addition, a new region was identified containing another arginine residue that appears to be conserved in all members of the family. To test further the importance of this newly identified arginine residue, mutants in the Cre protein from phase P1, a member of this integrase family, have been constructed which alter this residue. The mutations which change arginine to lysine and arginine to cysteine depress catalytic activity but not site-specific binding to the lox site. This result is expected for a conserved active site residue. This computer analysis also provides a means for searching for new members of the integrase family.

Amino Acid Sequence↗
PubMed1992

Identification of conserved amino acid residues critical for human immunodeficiency virus type 1 integrase function in vitro.

We have probed the structural organization of the human immunodeficiency virus type 1 integrase protein by limited proteolysis and the functional organization by site-directed mutagenesis of selected amino acid residues. A central region of the protein was relatively resistant to proteolysis. Proteins with altered amino acids in this region, or in the N-terminal part of the protein that includes a putative zinc-binding motif, were purified and assayed for 3' processing, DNA strand transfer, and disintegration activities in vitro. In general, these mutations had parallel effects on 3' processing and DNA strand transfer, suggesting that integrase may utilize a single active site for both reactions. The only proteins that were completely inactive in all three assays contained mutations at conserved amino acids in the central region, suggesting that this part of the protein may be involved in catalysis. In contrast, none of the mutations in the N-terminal region resulted in a protein that was inactive in all three assays, suggesting that this part of integrase may not be essential for catalysis. The disintegration reaction was particularly insensitive to these amino acid substitutions, indicating that some function that is important for 3' processing and DNA strand transfer may be dispensable for disintegration.

Amino Acid Sequence↗
PubMed2026

Cre-loaded integrase-defective lentiviral vectors for targeted cassette exchange in CHO cells.

Genome-modifying enzymes, such as recombinases and CRISPR-associated nucleases, enable targeted gene insertion when delivered transiently to minimize off-target effects. Precise genome engineering requires controlled enzyme activity, as well as efficient donor DNA transfer. Integrase-defective lentiviral vectors (IDLVs) provide a promising platform for transient episomal DNA transfer; however, their integration efficiency depends on complementary genome-targeting strategies. Here, we engineered Cre-loaded IDLVs (Cre-IDLVs) that co-package lentiviral vector genomes together with bioactive Cre recombinase. Cre was inserted into the Gag region of an integrase-defective gag-pol construct, allowing for efficient encapsidation and protease-mediated release during virion maturation without compromising the viral titer. The resulting particles carried donor cassettes flanked by heterospecific loxP sites. When applied to CHO founder cells harboring compatible genomic loxP landing pads, Cre-IDLVs efficiently mediated recombination-mediated cassette exchange, producing the highest number of G418-resistant colonies among the plasmid ratios tested. Genomic PCR and sequencing confirmed precise locus-specific insertion without detectable random integration in the analyzed clones. These findings establish Cre-IDLVs as a streamlined dual-delivery platform that couples transient recombinase activity with episomal donor DNA transfer. This hybrid lentiviral strategy provides a programmable approach for controlled and site-specific genome modification in mammalian cells.

Integrases↗
PubMed1988

Structural and functional analysis of Tn4430: identification of an integrase-like protein involved in the co-integrate-resolution process.

The 4149-bp transposon Tn4430 from Bacillus thuringiensis is delineated by 38-bp inverted repeats and codes for a 113-kd protein that shares homology with the transposases (TnpA) of Tn3, Tn21 and Tn501. Through transpositional recombination, this protein generates the formation of co-integrates between both donor and target replicons, with duplication of Tn4430 molecules. These features are characteristic of transposons of the Tn3 family (class II elements). The second step of the transposition process, the co-integrate resolution, is mediated by a 32-kd protein. This protein (TnpI) displays regional similarities with site-specific recombinases of the integrase family, such as Int of bacteriophage lambda, Cre of bacteriophage P1 or TnpA and TnpB of the Tn554 transposon. Moreover, the 250-bp sequence upstream to the tnpI gene contains several structural features that are reminiscent of the attP attachment site of phage lambda. This unique association between the integrase-like TnpI recombinase and the TnpA transposase qualifies Tn4430 as a member of a new group within the class II mobile genetic elements.

Amino Acid Sequence↗
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↗