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At least 1,189 records · Page 66Linked to original sources
PubMed1994

Xer-mediated site-specific recombination at cer generates Holliday junctions in vivo.

Normal segregation of the Escherichia coli chromosome and stable inheritance of multicopy plasmids such as ColE1 requires the Xer site-specific recombination system. Two putative lambda integrase family recombinases, XerC and XerD, participate in the recombination reactions. We have constructed an E. coli strain in which the expression of xerC can be tightly regulated, thereby allowing the analysis of controlled recombination reactions in vivo. Xer-mediated recombination in this strain generates Holliday junction-containing DNA molecules in which a specific pair of strands has been exchanged in addition to complete recombinant products. This suggests that Xer site-specific recombination utilizes a strand exchange mechanism similar or identical to that of other members of the lambda integrase family of recombination systems. The controlled in vivo recombination reaction at cer requires recombinase and two accessory proteins, ArgR and PepA. Generation of Holliday junctions and recombinant products is equally efficient in RuvC- and RuvC+ cells, and in cells containing a multicopy RuvC+ plasmid. Controlled XerC expression is also used to analyse the efficiency of recombination between variant cer sites containing sequence alterations and heterologies within their central regions.

Aminopeptidases↗
PubMed2006

Anti-human immunodeficiency virus activity of 3,4,5-tricaffeoylquinic acid in cultured cells of lettuce leaves.

3,4,5-Tricaffeoylquinic acid (TCQA) that is not found in intact plant of lettuce leaves was isolated from the cultured cells. The intact plant produced chicoric acid (dicaffeoyl tartaric acid: L-CCA) as well as chlorogenic acid (3-caffeoylquinic acid: 3-CQA) as the major metabolites. After subculturing of the cells for 40 days, the amount of 3,4,5-TCQA reached to 0.14 mg/g fresh weight. The inhibitory effect of 3,4,5-TCQA for human immunodeficiency virus (HIV) Type 1 integrase was assayed. Anti-HIV activity using HIV and MT-2 cells was 1.15 microM and IC(50) against HIV integrase was 0.063 microM whereas cell toxicity of this chemical was expressed as 5% death of all living cells to be 18.4 microM. The HIV inhibitory effect of 3,4,5-TCQA was the highest in values among L-CCA, and other dicaffeoylquinic acids. This data will provide a new possibility for creating a new drug design for HIV.

Anti-HIV Agents↗
PubMed1995

Characterization of the interaction between the lambda intasome and attB.

Bacteriophage lambda DNA integrates into the chromosome of Escherichia coli by first forming an intasome at the phage attachment site on the phage DNA with the integrase Int and integration host factor. This intasome searches the host chromosome for the bacterial attachment site (attB) and then orchestrates two sequential strand exchange reactions to achieve integration. This study characterizes the weak interaction of the intasome and attB. The hypothesis that all of the proteins necessary for integration are brought to the reaction site by the intasome is given additional support by showing that the concentration of phage attachment site and not attB determines the optimal concentration of proteins for integration. The value of the dissociation constant of the complex formed between the intasome and attB is determined in two different ways. First, the rate of the integration reaction is measured as a function of the attB DNA concentration. The saturation constant reflects the dissociation constant of the complex. Second, a recombination reaction is inhibited by the introduction of varying amounts of a second attB with a sequence change that blocks recombination with this site. The inhibition constant reflects the dissociation constant of the intasome and altered attB in this experiment. The two methods agree and give a dissociation constant of approximately 300 nM. attB contains two core binding sites for the intasome; it is shown that both are necessary for efficient capture. The value of the dissociation constants are considerably lower when a mutant integrase, IntE174K, is used. This increased affinity for core sites can explain how IntE174K can function in the absence of integration host factor. The inhibition constants also show dependence on the exact sequence of the inhibiting attB. Possible implications of this dependence are discussed.

Bacterial Proteins↗
PubMed1997

Characterization of the mycobacteriophage L5 attachment site, attP.

Lysogenization of mycobacteriophage L5 involves integration of the phage genome into the Mycobacterium smegmatis chromosome. Integration occurs by a site-specific recombination event between a phage attachment site, attP, and a bacterial attachment site, attB, which is catalyzed by the phage-encoded integrase protein. DNase I footprinting reveals that L5 integrase binds to two types of sites within attP which span an unexpectedly large region of 413 bp: seven arm-type sites (P1 to P7) each of which correspond to a consensus sequence 5'-TGCaaCtcYy, and core-type sites at the points of strand exchange. Mutational analyses indicate that not all of the arm-type sites are required for integration, and that the P3 site and the rightmost pair of sites (P6 and P7) are dispensable for integration. We show that a 252 bp segment of attP DNA is sufficient for efficient integrative recombination and that int can be provided in trans for simple and efficient transformation of the mycobacteria.

Bacteriophages↗
PubMed2000

Major groove recognition by three-stranded beta-sheets: affinity determinants and conserved structural features.

We present the results of a rational mutagenesis and binding-affinity study of the three-stranded beta-sheet-DNA interface in the complex formed by the amino-terminal DNA-binding domain of the Tn916 integrase protein and its cognate binding site. The relative importance of interfacial contacts present in its NMR-derived solution structure have been tested through mutagenesis, fluorescence anisotropy, and intrinsic quenching DNA-binding assays. We find that seven protein-DNA hydrogen bonds (two base-specific and five to phosphate groups) significantly contribute to the level of affinity. These interactions span the entire DNA-binding surface on the protein, but primarily originate from residues in only two strands of the sheet and loop L2. Interestingly, we show that highly populated, precisely defined intermolecular hydrogen bonds in the ensemble of conformers are invariably important for DNA-binding, implying that NMR-derived solution structures provide direct insight into the energetics of recognition. Unusual three-stranded beta-sheet-DNA interfaces have recently been discovered in three unrelated protein-DNA complexes. A comparative analysis of these structures reveals similar sheet positioning, the presence of two invariant interfacial contacts to the phosphodiester backbone, and two semi-conserved base-specific hydrogen bonds. Two of these conserved contacts significantly contribute to the affinity of the integrase-DNA complex, suggesting that the three-stranded beta-sheet DNA-binding motif exhibits conserved principles of recognition.

Amino Acid Motifs↗
PubMed1993

SSV1-encoded site-specific recombination system in Sulfolobus shibatae.

We present evidence for the existence of a conservative site-specific recombination system in Archaea by demonstrating integrative recombination of Sulfolobus shibatae virus SSV1 DNA with the host chromosome, catalysed by the SSV1-encoded integrase in vitro. The putative int gene of SSV1 was expressed in Escherichia coli yielding a protein of about 39 kDa. This protein alone efficiently recombined linear DNA substrates containing chromosomal (attA) and viral (attP) attachment sites; recombination with either negatively or positively supercoiled SSV1 DNA was less efficient. Intermolecular attA x attA and attP x attP recombination was also promoted by the SSV integrase. The invariant 44 bp "common attachment core" present in all att sites contained sufficient information to allow recombination, whilst the flanking sequences effected the efficiency. These features clearly distinguish the SSV1--encoded site--specific recombination system from others and make it suitable for the study of regulatory mechanisms of SSV1 genome--host chromosome interaction and investigations of the evolution of the recombination machinery.

Bacteriophages↗
PubMed2002

Phylogeny derived from coding retroviral genome organization.

When divergence between viral species is large, the analysis and comparison of nucleotide or protein sequences are dependent on mutation biases and multiple substitutions per site leading, among other things, to the underestimation of branch lengths in phylogenetic trees. To avoid the problem of multiply substituted sites, a method not directly based on the nucleic or protein sequences has been applied to retroviruses. It consisted of asking questions about genome structure or organization, and gene function, the series of answers creating coded sequences analyzed by phylogenic software. This method recovered the principal retroviral groups such as the lentiviruses and spumaviruses and highlighted questions and answers characteristic of each group of retroviruses. In general, there was reasonable concordance between the coded genome methodology and that based on conventional phylogeny of the integrase protein sequence, indicating that integrase was fixing mutations slowly enough to marginalize the problem of multiple substitutions at sites. To a first approximation, this suggests that the acquisition of novel genetic features generally parallels the fixation of amino acid substitutions.

Genome, Viral↗
PubMed2004

The genome of BCJA1c: a bacteriophage active against the alkaliphilic bacterium, Bacillus clarkii.

The sequence of the genome of the first alkaliphilic bacteriophage has been determined. Temperate phage BCJA1 possesses a terminally redundant genome of approximately 41 kb, with a mol% G + C content of 41.7 and 59 genes arranged predominantly into two divergent transcriptons. The integrase gene of this phage is unique in that it contains a ribosomal slippage site. While this type of translational regulation occurs in the synthesis of transposase, this is the first time that it has been observed in a bacteriophage integrase. The DNA replication, recombination, packaging, and morphogenesis proteins show their greatest sequence similarity to phages and prophages from the genus Streptococcus. Host specificity, lysin, and lysogeny maintenance functions are most closely related to genes from Bacillus species.

Bacillus↗
PubMed1994

Inhibition of the in vitro integration of Moloney murine leukemia virus DNA by the DNA minor groove binder netropsin.

In search of potential inhibitors of integration of retroviral DNA into host cells genome, we have investigated the effect of the external DNA binder netropsin on the in vitro insertion of long terminal repeat (LTR) ends of Moloney murine leukemia virus (M.MuLV) as catalysed by integrase purified from baculovirus strain expression vector. In agreement with the preferential binding of netropsin to A+T rich sequences, footprinting experiments have shown that this drug selectively binds to the 5'-TTTCAT LTR end sequence which is included in the DNA binding site of integrase. This feature results in the potent inhibition of both reactions involved in the insertion process, namely, nucleolytic cleavage and strand transfer. The relation between netropsin binding to A+T rich region of M.MuLV LTR end and inhibition of insertion is strongly suggested from the inability of the drug to inhibit the insertion of HIV U3 LTR end which displays a G+C rich sequence. Selective inhibition of integration of viral DNA appears to be feasible using drugs recognizing LTR end sequences.

Amino Acid Sequence↗
PubMed1987

Integration of satellite bacteriophage P4 in Escherichia coli. DNA sequences of the phage and host regions involved in site-specific recombination.

We determined the DNA sequences of regions essential for bacteriophage P4 integration. A 20 base-pair core sequence in both phage (P4attP) and host (P4attB) attachment regions contains the recombination site. In P4attP this sequence is flanked by five repeated sequences. A 1.3 x 10(3) base open reading frame codes for P4 integrase. Two possible promoters are upstream from P4int. One would be recognized by Escherichia coli RNA polymerase and may be repressed by integrase protein. The second would be recognized by RNA polymerase modified after infection by a P4 helper phage, P2. The P4attB core sequence is the 3' end of a leucine tRNA gene. Downstream from this tRNA in E. coli K-12 is a region homologous to P4int that may be part of a cryptic prophage.

Bacteriophages↗
PubMed1992

Ulysses transposable element of Drosophila shows high structural similarities to functional domains of retroviruses.

We have determined the DNA structure of the Ulysses transposable element of Drosophila virilis and found that this transposon is 10,653 bp and is flanked by two unusually large direct repeats 2136 bp long. Ulysses shows the characteristic organization of LTR-containing retrotransposons, with matrix and capsid protein domains encoded in the first open reading frame. In addition, Ulysses contains protease, reverse transcriptase, RNase H and integrase domains encoded in the second open reading frame. Ulysses lacks a third open reading frame present in some retrotransposons that could encode an env-like protein. A dendrogram analysis based on multiple alignments of the protease, reverse transcriptase, RNase H, integrase and tRNA primer binding site of all known Drosophila LTR-containing retrotransposon sequences establishes a phylogenetic relationship of Ulysses to other retrotransposons and suggests that Ulysses belongs to a new family of this type of elements.

Amino Acid Sequence↗
PubMed1988

The DNA intermediate in yeast Ty1 element transposition copurifies with virus-like particles: cell-free Ty1 transposition.

Yeast Ty1 elements are retrotransposons that transpose via an RNA intermediate found in a virus-like particle (Ty-VLP). A Ty-encoded reverse transcriptase activity found inside the particles is capable of giving rise to full-length reverse transcripts. The predominant form of these reverse transcripts is a full-length linear duplex DNA. We have developed a cell-free system for transposition of Ty1 DNA molecules into a bacteriophage lambda target. Purified Ty-VLPs and target DNA are the only macromolecular components required for the transposition reaction. A TYB-encoded protein, p90-TYB, contains amino acid sequences that are similar to those of retroviral integrase proteins. Mutations in the integrase coding region abolish transposition both in vivo and in vitro.

Antibodies, Monoclonal↗
PubMed2004

HIV-chemotherapy and -prophylaxis: new drugs, leads and approaches.

In recent years, significant progress has been made towards the chemotherapy (and prophylaxis) of HIV infections. This progress is situated at three different levels. (i) New anti-HIV drugs have been approved for clinical use and have entered the market: the virus entry inhibitor enfuvirtide (Fuzeon), the nucleoside reverse transcriptase inhibitor (NRTI) emtricitabine (Emtriva), the nucleotide reverse transcriptase inhibitor (NtRTI) tenofovir disoproxil fumarate (Viread trade mark ) and the HIV protease inhibitor (PI) atazanavir (Reyataz trade mark ). (ii) Other compounds have proceeded through preclinical and/or clinical development: CXCR4 antagonists (i.e. AMD070), CCR5 antagonists (i.e. SCH-C), NRTIs (such as amdoxovir), NNRTIs (such as etravirine), integrase inhibitors (such as S-1360) and PIs (such as tipranavir). (iii) Yet other compounds, acting by novel mechanisms, have recently been identified as anti-HIV agents that seem worthy of further (pre)clinical development: cell receptor CD4 down-modulators (i.e. cyclotriazadisulfonamides), viral envelope gp120-binding agents such as plant lectins and glycopeptide antibiotics, HIV integrase inhibitors such as the pyranodipyrimidine V-165, and two new classes of compounds (i.e. N-aminoimidazoles and pyridine oxide derivatives) which seem to interfere with a post-integration, transcription transactivation event. Taken together, it is obvious that the approaches for the treatment of HIV infections in recent years have become both more diverse and more efficient.

Amino Acid Sequence↗
PubMed2006

Dynamic analysis of a genomic island in Magnetospirillum sp. strain AMB-1 reveals how magnetosome synthesis developed.

The entire structure of a 98 kb genomic region that abounds in genes related to magnetosome synthesis was first described in the Magnetospirillum sp. strain AMB-1. The deletion of this 98 kb genomic region and the circular form after excision from the chromosome was detected by PCR amplification. This strongly suggests that the region has undergone a lateral gene transfer. The region has the characteristics of a genomic island: low GC content, location between two repetitive sequences, and the presence of an integrase in the flanking region of the first repetitive sequence. This 98 kb genomic region has the potential for transfer by the integrase activity. Comparative genome analysis revealed other regions with a high concentration of orthologs in magnetic bacteria besides the 98 kb region, and magnetosome synthesis seemed to need not only the exogenous 98 kb region, but also other orthologs and individually originating genes.

Gene Transfer Techniques↗
PubMed2004

The Yersinia high-pathogenicity island (HPI): evolutionary and functional aspects.

The high-pathogenicity island (HPI) is a genomic island essential for the mouse-virulence phenotype in Yersinia and indispensable for pathogenicity of Yersinia and certain pathotypes of Escherichia coli. In contrast to most genomic islands, the HPI is a functional island widely disseminated among members of the family of Enterobacteriaceae. The HPI-encoded phage P4-like integrase together with excisionase and recombination sites make up the genetic mobility module of the island, while the siderophore yersiniabactin biosynthesis and uptake system comprises its functional part with respect to fitness and pathogenicity. The HPI-integrase promotes integration of the island into attB sites represented by three to four asn tDNAs in Yersinia pestis and E. coli. An additional enzyme, excisionase, is essential for efficient excision of the HPI from the initial site of integration. Furthermore a unique type of HPI has been characterized in the E. coli strain ECOR31 carrying a functional conjugative mating pair formation (Mpf) and a DNA-processing system, both of which are characteristic of integrative and conjugative elements (ICE). A model of conjugative transfer for the dissemination of HPIs is proposed in which the excised HPI is mobilized to a new recipient either trapped by a transmissive asn tDNA-carrying plasmid or autonomously as an ICE named ICEEcl.

Animals↗
PubMed2005

Receipt of the C-terminal tail from a neighboring lambda Int protomer allosterically stimulates Holliday junction resolution.

Bacteriophage lambda integrase (Int) catalyzes the integration and excision of the phage lambda chromosome into and out of the Esherichia coli host chromosome. The seven carboxy-terminal residues (C-terminal tail) of Int comprise a context-sensitive regulatory element that links catalytic function with protein multimerization and also coordinates Int functions within the multimeric recombinogenic complex. The experiments reported here show that the beta5-strand of Int is not simply a placeholder for the C-terminal tail but rather exerts its own allosteric effects on Int function in response to the incoming tail. Using a mutant integrase in which the C-terminal tail has been deleted (W350ter), we demonstrate that the C-terminal tail is required for efficient and accurate resolution of Holliday junctions by tetrameric Int. Addition of a free heptameric peptide of the same sequence as the C-terminal tail partially reverses the W350ter defects by stimulating Holliday junction resolution. The peptide also stimulates the topoisomerase function of monomeric W350ter. Single residue alterations in the peptide sequence and a mutant of the beta5 strand indicate that the observed stimulation arises from specific contacts with the beta5 strand (residues 239-243). The peptide does not stimulate binding of W350ter to its cognate DNA sites and therefore appears to recapitulate the effects of the normal C-terminal tail intermolecular contacts in wild-type Int. Models for the allosteric stimulation of Int activity by beta5 strand contacts are discussed.

Allosteric Site↗
PubMed2005

Molecular tools to detect the IncJ elements: a family of integrating, antibiotic resistant mobile genetic elements.

The IncJ group of enterobacterial mobile genetic elements, which include R391, R392, R705, R997 and pMERPH, have been shown to be site-specific integrating elements encoding variable antibiotic and heavy metal resistance genes. They insert into a specific 17-bp site located in the prfC gene, encoding peptide release factor 3, in Escherichia coli and other hosts. A key feature of known IncJ elements is the presence of a site-specific recombination module consisting of an attachment site on the element and an integrase-encoding gene of the tyrosine recombinase class, which promotes integration between the attachment site on the element and a similar site on the host chromosome. We have cloned and sequenced the integrases from a number of known IncJ elements and designed PCR primers for specific amplification of this gene. Using conserved regions of enterobacterial prfC genes upstream and downstream of the insertion site, and conserved sequences at the ends of the integrated IncJ elements, we have designed specific primers to amplify across the integrated IncJ attL and attR junction fragments. Alignment of over 30 enterobacterial prfC-like genes indicates that the primers designed to amplify attR junction would amplify IncJ element: host junctions from a wide variety of hosts. The IncJ elements have been shown to sensitise recA(+)E. coli K12 strains to UV irradiation. A simple and rapid procedure for demonstrating this effect is described. These tools should enable the rapid detection of such elements in clinical and environmental settings.

Attachment Sites, Microbiological↗
PubMed2003

Sequence of the loxP site determines the order of strand exchange by the Cre recombinase.

Conservative site-specific recombinases of the integrase family carry out recombination via a Holliday intermediate. The Cre recombinase, a member of the integrase family, was previously shown to initiate recombination by cleaving and exchanging preferentially on the bottom strand of its loxP target sequence. We have confirmed this strand bias for an intermolecular recombination reaction that used wild-type loxP sites and Cre protein. We have examined the sequence determinants for this strand preference by selectively mutating the two asymmetric scissile base-pairs in the lox site (those immediately adjacent to the sites of cleavage by Cre). We found that the initial strand exchange occurs preferentially next to the scissile G residue. Resolution of the Holliday intermediate thus formed takes place preferentially next to the scissile A residue. Lys86, which contacts the scissile nucleotides in the Cre-lox crystal structures, was important for establishing the strand preference in the resolution of the loxP-Holliday intermediate, but not for the initiation of recombination between loxP sites.

Base Pairing↗