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PubMed1995

Comparative studies of bacterially expressed integrase proteins of caprine arthritis-encephalitis virus, maedi-visna virus and human immunodeficiency virus type 1.

Integrase (IN) proteins mediate an essential step in retroviral life cycles, the integration of reverse-transcribed viral DNA into the host genome. To create tools for direct comparative investigations, hexahistidine-tagged IN proteins of the phylogenetically related lentiviruses caprine arthritis-encephalitis virus (CAEV), maedi-visna virus (MVV) and human immunodeficiency virus type 1 (HIV-1) were expressed in Escherichia coli. After purification by affinity chromatography, the active enzymes were compared in vitro for their site-specific cleavage, integration and disintegration activities on cognate and non-cognate oligonucleotide substrates. It was found that CAEV IN and MVV IN catalyse both site-specific cleavage and disintegration with high efficiencies, reduced substrate specificities and similar reaction patterns. Comparisons with the respective activities of HIV-1 IN revealed basic functional similarities as well as considerable differences such as more restricted substrate requirements for site-specific cleavage. On the other hand, all three enzymes catalyse disintegration almost independent of the substrate origin. Furthermore, MVV IN was shown to join oligonucleotides as efficiently as HIV-1 IN, albeit with reduced substrate specificity. In contrast, no detectable strand transfer activities occurred with CAEV IN.

Amino Acid Sequence↗
PubMed1999

An active foamy virus integrase is required for virus replication.

Foamy viruses (FVs) make use of a replication strategy which is unique among retroviruses and shows analogies to hepadnaviruses. The presence of an integrase (IN) and obligate provirus integration distinguish retroviruses from hepadnaviruses. To clarify whether a functional IN is required for FV replication, a mutant in the highly conserved DD35E motif of the active centre was analysed. This mutant was found to be able to express Gag and Pol protein precursors and cleavage products and to generate and deliver cDNA. However, this mutant was replication-deficient. The junctions of individual foamy proviruses with cellular DNA were sequenced. The findings suggest that FV integration is asymmetrical, because the proviruses started with what is believed to be the U3 end of the free linear DNA to generate the conventional TG dinucleotide, while apparently two nucleotides from the U5 end were cleaved to create the complementary CA dinucleotide. Alignment of known FV genome sequences indicated that this mechanism of integration is not restricted to the two FV isolates from which integrates were studied, but appears to be a common feature of this retrovirus subfamily. In conclusion, with respect to the necessity of a functionally active IN for virus replication FVs behave like other retroviruses; their mechanism of integration, however, is probably unique.

Animals↗
PubMed2003

Development of the Micromonospora carbonacea var. africana ATCC 39149 bacteriophage pMLP1 integrase for site-specific integration in Micromonospora spp.

Micromonospora carbonacea var. africana ATCC 39149 contains a temperate bacteriophage, pMLP1, that is present both as a replicative element and integrated into the chromosome. Sequence analysis of a 4.4 kb KpnI fragment revealed pMLP1 att/int functions consisting of an integrase, an excisionase and the phage attachment site (attP). Plasmids pSPRH840 and pSPRH910, containing the pMLP1 att/int region, were introduced into Micromonospora spp. by conjugation from Escherichia coli. Sequence analysis of DNA flanking the integration site confirmed site-specific integration into a tRNAHis gene in the chromosome. The pMLP1 attP element and chromosomal bacterial attachment (attB) site contain a 24 bp region of sequence identity located at the 3' end of the tRNA. Integration of pMLP1-based plasmids in M. carbonacea var. africana caused a loss of the pMLP1 phage. Placement of an additional attB site into the chromosome allowed integration of pSPRH840 into the alternate attB site. Plasmids containing the site-specific att/int functions of pMLP1 can be used to integrate genes into the chromosome.

Attachment Sites, Microbiological↗
PubMed2005

Yeast system as a model to study Moloney murine leukemia virus integrase: expression, mutagenesis and search for eukaryotic partners.

Moloney murine leukemia virus (M-MuLV) integrase (IN) catalyses the insertion of the viral genome into the host chromosomal DNA. The limited solubility of the recombinant protein produced in Escherichia coli led the authors to explore the use of Saccharomyces cerevisiae for expression of M-MuLV IN. IN was expressed in yeast and purified by chromatography on nickel-NTA agarose. IN migrated as a single band in SDS-PAGE and did not contain IN degradation products. The enzyme was about twofold more active than the enzyme purified from E. coli and was free of nucleases. Using the yeast system, the substitution of the putative catalytic amino acid Asp184 by alanine was also analysed. The mutated enzyme was inactive in the in vitro assays. This is the first direct demonstration that mutation of Asp184 inactivates M-MuLV IN. Finally, S. cerevisiae was used as a model to assess the ability of M-MuLV IN to interact with eukaryotic protein partners. The expression of an active M-MuLV IN in yeast strains deficient in RAD52 induced a lethal effect. This phenotype could be attributed to cellular damage, as suggested by the viability of cells expressing inactive D184A IN. Furthermore, when active IN was expressed in a yeast strain lacking the ySNF5 transcription factor, the lethal effect was abolished, suggesting the involvement of ySNF5 in the cellular damage induced by IN. These results indicate that S. cerevisiae could be a useful model to study the interaction of IN with cellular components in order to identify potential counterparts of the natural host.

Animals↗
PubMed2006

Expression of phage P4 integrase is regulated negatively by both Int and Vis.

Phage P4 int gene encodes the integrase responsible for phage integration into and excision from the Escherichia coli chromosome. Here, the data showing that P4 int expression is regulated in a complex manner at different levels are presented. First of all, the Pint promoter is regulated negatively by both Int and Vis, the P4 excisionase. The N-terminal portion of Int appears to be sufficient for such a negative autoregulation, suggesting that the Int N terminus is implicated in DNA binding. Second, full-length transcripts covering the entire int gene could be detected only upon P4 infection, whereas in P4 lysogens only short 5'-end covering transcripts were detectable. On the other hand, transcripts covering the 5'-end of int were also very abundant upon infection. It thus appears that premature transcription termination and/or mRNA degradation play a role in Int-negative regulation both on the basal prophage transcription and upon infection. Finally, comparison between Pint-lacZ transcriptional and translational fusions suggests that Vis regulates Int expression post-transcriptionally. The findings that Vis is also an RNA-binding protein and that Int may be translated from two different start codons have implications on possible regulation models of Int expression.

Artificial Gene Fusion↗
PubMed1995

Enhanced and coordinated processing of synapsed viral DNA ends by retroviral integrases in vitro.

We have designed novel substrates to investigate the first step in retroviral integration: the site-specific processing of two nucleotides from the 3' ends of viral DNA. The substrates consist of short duplex oligodeoxynucleotides whose sequences match those of the U3 and U5 ends of viral DNA but are covalently synapsed across the termini by short, single-strand nucleotide linkers. We show here that the optimal separation between termini in a synapsed-end substrate for avian sarcoma/leukosis virus (ASV) IN is 2 nucleotides. This places the two conserved 5'-CA-3' processing sites 6 nucleotides apart, a separation equal to the staggered cut in target DNA produced by this enzyme during the subsequent joining reaction. Based on estimates of initial reaction rates, this synapsed-end substrate is processed by IN at > 10-fold higher efficiency than observed with an equivalent mixture of U3 and U5 single-end (uncoupled) substrates. Enhanced processing is maintained at low IN concentrations, suggesting that the synapsed-end substrate may facilitate enzyme multimerization. Enhanced processing by HIV-1 IN, which produces a 5-bp stagger during integration, was observed with a synapsed-end substrate in which the separation between processing sites was 5 nucleotides. These observations provide estimates of the distances between active sites in the multimeric IN-DNA complexes of ASV and HIV-1. Our results also show that processing of paired U3 and U5 ends need not be coupled temporally. Finally, we observed that substrates that paired a wild-type with a mutated terminus were cleaved poorly at both ends. Thus, in vitro processing of the synapsed-end substrates requires specific recognition of the sequences at both ends. These findings provide new insights into the mechanism of integrative recombination by retroviral integrases and, by extension, other prokaryotic and eukaryotic transposases that are related to the viral enzymes.

Avian Sarcoma Viruses↗
PubMed2005

A non-natural dinucleotide containing an isomeric L-related deoxynucleoside: dinucleotide inhibitors of anti-HIV integrase activity.

The first X-ray crystal structure of a non-natural dinucleotide, 5'-O-phosphoryl-1'-deoxy-2'-isoadenylyl-(3' --> 5')-cytidine 6.5-hydrate (pIsodApC), C19H26N8O13P2 x 6.5H2O, belonging to a family of dinucleotides that contain an isomeric nucleoside component, is described. A complex system of hydrogen bonds between water molecules and various sites on the dinucleotide was found. All H atoms were located from electron-density difference maps, which allowed identification of protonation sites. Compounds of this family have been found to bind at the active site of HIV integrase and to be inhibitors of this key viral enzyme. These dinucleotides are completely resistant to cleavage by exonucleases; an abnormal dihedral angle twist in an internucleotide phosphate bond revealed in the X-ray crystal structure may be contributing to this unusual stability towards nucleases.

Crystallography, X-Ray↗
PubMed1999

Purification, solution properties and crystallization of SIV integrase containing a continuous core and C-terminal domain.

The C-terminal two-thirds segment of integrase derived from the simian immunodeficiency virus has been cloned, expressed in Escherichia coli, and purified to greater than 95% homogeneity. The protein encompasses amino-acid residues 50-293 and contains a F185H substitution to enhance solubility. In dilute solutions at concentrations below 1 mg ml(-1), the enzyme is predominantly dimeric. At the higher concentrations (>10 mg ml(-1)) required to enable crystallization, the enzyme self-associates to form species with molecular weights greater than 200 kDa. Despite the apparent high aggregation in solution, the enzyme crystallizes from a 8%(v/v) polyethylene glycol (molecular weight 6000) solution in a form suitable for X-ray diffraction studies. The resulting single crystals belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 79.76, b = 99.98, c = 150.2 A, alpha = beta = gamma = 90 degrees and Z = 4. Under X-ray irradiation generated with a rotating-anode generator, the crystals diffract to 2.8 A resolution and allow collection of a native 3 A resolution diffraction data set.

Cloning, Molecular↗
PubMed2003

Protein folding coupled to DNA binding in the catalytic domain of bacteriophage lambda integrase detected by mass spectrometry.

Bacteriophage lambda integrase (lambda-Int) is the prototypical member of a large family of enzymes that catalyze site-specific DNA recombination via single-strand cleavage and the formation of a Holliday junction intermediate. Crystallographic and biochemical evidence indicate that substantial conformational change (i.e., folding) in the catalytic domain of the protein is required for substrate recognition and catalysis. We have examined the solution conformation of the catalytic domain (C170) in the absence and presence of a cognate "half-site" DNA oligonucleotide by electrospray ionization mass spectrometry, and circular dichroism and fluorescence spectroscopy. The distribution of ions in the positive ion electrospray mass spectrum of the free protein reveals the presence of three distinct species in solution, one corresponding to the folded protein, one to the unfolded protein, and one to a dimer. In the presence of DNA, ions are observed only for the protein-DNA complex and the folded form of the free protein. We therefore conclude that DNA binding stabilizes the global fold of the protein in a manner that is consistent with folding-coupled target recognition as a mechanism to control site-specific recombination. Furthermore, we find that inspection of the charge state distribution of ions in electrospray mass spectra provides a quick and effective means to identify conformational heterogeneity of proteins in solution and to investigate dynamic protein-nucleic acid interactions.

Amino Acid Sequence↗
PubMed1993

Secondary-sites for integration mediated by the Tn21 integrase.

The integrase encoded by the integron of the transposon Tn21 can mediate the site-specific fusion of two plasmids if there is a recombination hot spot (59 bp element) in one of them and the sequence GWTMW in the other. The use of this latter, loosely defined site explains how antibiotic-resistance genes could first become associated with integrons.

Base Sequence↗
PubMed2004

Unequal access of chromosomal regions to each other in Salmonella: probing chromosome structure with phage lambda integrase-mediated long-range rearrangements.

We have investigated the fluidity of the Salmonella chromosome architecture using the phage lambda site-specific recombination system as a probe. We determined how chromosome position affects the extent of integrase-mediated recombination between pairs of inversely oriented att sites at various loci. We also investigated the accessibility of each chromosomal att site to an extrachromosomal partner carried on a low-copy plasmid. Recombination events were assayed by semi-quantitative polymerase chain reaction of the attP product. The extent of recombination between the chromosome and the plasmid was generally higher than intrachromosomal recombination except for two loci, araA::attL and galT::attL, which gave no detectable recombination with any other locus. Based on 20 intervals, we found that chromosomal locations are not equally accessible to each other. Although multiple factors probably affect accessibility, the most important is the specific combination of the end-points used. Neither the size of the intervals nor the accessibility of individual end-points to extrachromosomal sequences is as important. These results suggest that the chromosome is not completely fluid but rather organized in some way, with barriers that limit the movement of DNA within the cell. The nature of the barriers involved in chromosomal organization remains to be determined.

Attachment Sites, Microbiological↗
PubMed2006

Characterization of a conjugative transposon integrase, IntDOT.

Sequence analysis revealed that the integrase of the Bacteroides conjugative transposon CTnDOT (IntDOT) might be a member of the tyrosine recombinase family because IntDOT has five of six highly conserved residues found in the catalytic domains of tyrosine recombinases. Yet, IntDOT catalyses a reaction that appears to differ in some respects from well-studied tyrosine recombinases such as that of phage lambda. To assess the importance of the conserved residues, we changed residues in IntDOT that align with conserved residues in tyrosine recombinases. Some substitutions resulted in a complete loss or significant decrease of integration activity in vivo. The ability of the mutant proteins to cleave and ligate CTnDOT attachment site (attDOT) DNA in vitro in general paralleled the in vivo results, but the H345A mutant, which had a wild-type level of integration in vivo, exhibited a slightly lower level of cleavage and ligation in vitro. Our results confirm the hypothesis that IntDOT belongs to the tyrosine recombinase family, but the catalytic core of the protein seems to have somewhat different organization. Previous DNA sequence analyses showed that CTnDOT att sites contain 5 bp non-homologous coupling sequences which were assumed to define the putative staggered sites of cleavage. However, cleavage assays showed that one of the cleavage sites is 2 bp away from the junction of CTnDOT and coupling sequence DNA. The site is in a region of homology that is conserved in CTnDOT att sites.

Bacterial Proteins↗
PubMed2006

Characterization of the integrase of NBU1, a Bacteroides mobilizable transposon.

NBU1 is a Bacteroides mobilizable transposon (MTn) that is integrated within the host chromosome and requires CTnDOT functions for its excision and transfer into a new host. The NBU1 integrase IntN1 has been classified as a tyrosine recombinase based on the presence of conserved residues. We created alanine mutants of the residues R291, K314, H393, R396, H419 and the conserved substitution Y429F and tested them for integration efficiency. The results suggest that these residues in IntN1 are important for integration, and Y429 could be the catalytic nucleophile. We employed suicide substrates and partially purified IntN1 to determine the positions of IntN1 cleavage within the 14 bp common core region that is identical in both NBU1 att sites. We show that IntN1 makes 7 bp staggered cuts on the top and bottom strands. From previous mutational analysis of the att sites, we show that two specific mutations near the site of bottom strand cleavage within this 7 bp region increased integration, and mutations of the two bases near top strand cleavage site had no effect on integration. These results indicate that IntN1 lacks the strict requirement for homology between the recombining sites seen with other tyrosine recombinases. We also show that phosphorothioate substitutions at the cleavage site and 1 bp downstream inhibited cleavage by IntN1. This differs from other studied tyrosine recombinases where inhibition occurs by substitutions at the cleavage site only.

Amino Acid Sequence↗
PubMed2006

Excision and transfer of the Mesorhizobium loti R7A symbiosis island requires an integrase IntS, a novel recombination directionality factor RdfS, and a putative relaxase RlxS.

The Mesorhizobium loti strain R7A symbiosis island is an Integrative Conjugative Element (ICE), herein termed ICEMlSymR7A, which integrates into a phetRNA gene. Integration reconstructs the phetRNA gene at one junction with the core chromosome, and a direct repeat of the 3-prime 17 bp of the gene is formed at the other junction. We show that the ICEMlSymR7AintS gene, which encodes an integrase of the phage P4 family, is required for integration and excision of the island. Excision also depended on a novel recombination directionality factor encoded by msi109 (rdfS). Constitutive expression of rdfS resulted in curing of ICEMlSymR7A. The rdfS gene is part of an operon with genes required for conjugative transfer, allowing co-ordinate regulation of ICEMlSymR7A excision and transfer. The excised form of ICEMlSymR7A was detectable during exponential growth but occurred at higher frequency during stationary phase. ICEMlSymR7A encodes homologues of the traR and traI genes of Agrobacterium tumefaciens that regulate Ti plasmid transfer via quorum sensing. The presence of a plasmid with cloned island traR traI2 genes resulted in excision of ICEMlSymR7A in all cells regardless of culture density, indicating that excision may be similarly regulated. Maintenance of ICEMlSymR7A in these cells depended on msi106 (rlxS) that encodes a putative relaxase. Transfer of the island to non-symbiotic mesorhizobia required intS, rlxS and rdfS. The rdfS and rlxS genes are conserved across a diverse range of alpha-, beta- and gamma-proteobacteria and identify a large family of genomic islands with a common transfer mechanism.

Amino Acid Sequence↗
PubMed2004

A novel approach to plastid transformation utilizes the phiC31 phage integrase.

Thus far plastid transformation in higher plants has been based on incorporation of foreign DNA in the plastid genome by the plastid's homologous recombination machinery. We report here an alternative approach that relies on integration of foreign DNA by the phiC31 phage site-specific integrase (INT) mediating recombination between bacterial and phage attachment sites (attB and attP, respectively). Plastid transformation by the new approach depends on the availability of a recipient line in which an attB site has been incorporated in the plastid genome by homologous recombination. Plastid transformation involves insertion of an attP vector into the attB site by INT and selection of transplastomic clones by selection for antibiotic resistance carried in the attP plastid vector. INT function was provided by either expression from a nuclear gene, which encoded a plastid-targeted INT, or expressing INT transiently from a non-integrating plasmid in plastids. Transformation was successful with both approaches using attP vectors with kanamycin resistance or spectinomycin resistance as the selective marker. Transformation efficiency in some of the stable nuclear INT lines was as high as 17 independently transformed lines per bombarded sample. As this system does not rely on the plastid's homologous recombination machinery, we expect that INT-based vectors will make plastid transformation a routine in species in which homologous recombination rarely yields transplastomic clones.

Attachment Sites, Microbiological↗
PubMed1997

The Kw recombinase, an integrase from Kluyveromyces waltii.

Site-specific recombinases of the integrase family share limited amino-acid-sequence similarity, but use a common reaction mechanism to recombine distinct DNA target sites. Here we report the characterisation of the Kw site-specific recombinase, encoded on the 2 mu-like plasmid pKWS1 from the yeast Kluyveromyces waltii. Using in vitro-translated Kw recombinase, we show that the protein is able to bind and to recombine its putative DNA target site. Recombination is conservative and the Kw target site has a spacer of seven base pairs. We show that Kw recombinase is able to mediate recombination in a mammalian cell line, thus, it has potential for use as a tool for genomic manipulation in heterologous systems.

Cell Line↗
PubMed1989

DNA looping generated by DNA bending protein IHF and the two domains of lambda integrase.

The multiprotein-DNA complexes that participate in bacteriophage lambda site-specific recombination were used to study the combined effect of protein-induced bending and protein-mediated looping of DNA. The protein integrase (Int) is a monomer with two autonomous DNA binding domains of different sequence specificity. Stimulation of Int binding and cleavage at the low affinity core-type DNA sites required interactions with the high affinity arm-type sites and depended on simultaneous binding of the sequence-specific DNA bending protein IHF (integration host factor). The bivalent DNA binding protein is positioned at high affinity sites and directed, by a DNA bending protein, to interactions with distant lower affinity sites. Assembly of this complex is independent of protein-protein interactions.

Bacterial Proteins↗
PubMed1994

Major antigenic region on the integrase (IN) protein of human immunodeficiency virus type 1 determined by reactivity of human sera and a monoclonal antibody to IN protein.

The gene encoding the integrase (IN) protein of human immunodeficiency virus type 1 (HIV-1) was expressed in vaccinia virus and Escherichia coli, and sera from 55 HIV-1-infected individuals were examined for immunoreactivity to the recombinant IN proteins by Western immunoblot. Approximately 98% (54 of 55) of the HIV-1-infected individuals showed reactivity to both the full-length IN protein of 32 kDa (IN32 protein) and the carboxy-terminal portion of the IN protein (IN17 protein). Serum samples from only 6 of the 54 antibody-positive individuals and a monoclonal antibody against the IN protein, 6F4, reacted with the amino-terminal portion of the IN protein (IN15 protein). The eight AIDS patients tested were seronegative to IN15 protein. The magnitude of reactivity to the recombinant IN proteins decreased slightly in the progression of the course of HIV-1 infection. These results suggest that a B-cell immunodominant epitope(s) on the IN protein is located on the C-terminal IN17 portion and that a minor epitope(s) recognizable by 6F4 and by rare patients is on the N-terminal IN15 portion.

Amino Acid Sequence↗