The Experts below are selected from a list of 24615 Experts worldwide ranked by ideXlab platform
Yves Pommier - One of the best experts on this subject based on the ideXlab platform.
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hiv 1 Integrase strand transfer inhibitors with reduced susceptibility to drug resistant mutant Integrases
ACS Chemical Biology, 2016Co-Authors: Xue Zhi Zhao, Christophe Marchand, Yves Pommier, Peter Cherepanov, Mathieu Metifiot, Steven J Smith, D P Maskell, Valerie E Pye, Katherine Fesen, Stephen H HughesAbstract:HIV Integrase (IN) strand transfer inhibitors (INSTIs) are among the newest anti-AIDS drugs; however, mutant forms of IN can confer resistance. We developed noncytotoxic naphthyridine-containing INSTIs that retain low nanomolar IC50 values against HIV-1 variants harboring all of the major INSTI-resistant mutations. We found by analyzing crystal structures of inhibitors bound to the IN from the prototype foamy virus (PFV) that the most successful inhibitors show striking mimicry of the bound viral DNA prior to 3′-processing and the bound host DNA prior to strand transfer. Using this concept of “bi-substrate mimicry,” we developed a new broadly effective inhibitor that not only mimics aspects of both the bound target and viral DNA but also more completely fills the space they would normally occupy. Maximizing shape complementarity and recapitulating structural components encompassing both of the IN DNA substrates could serve as a guiding principle for the development of new INSTIs.
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resistance to Integrase inhibitors
Viruses, 2010Co-Authors: Mathieu Metifiot, Christophe Marchand, Kasthuraiah Maddali, Yves PommierAbstract:Integrase (IN) is a clinically validated target for the treatment of human immunodeficiency virus infections and raltegravir exhibits remarkable clinical activity. The next most advanced IN inhibitor is elvitegravir. However, mutant viruses lead to treatment failure and mutations within the IN coding sequence appear to confer cross-resistance. The characterization of those mutations is critical for the development of second generation IN inhibitors to overcome resistance. This review focuses on IN resistance based on structural and biochemical data, and on the role of the IN flexible loop i.e., between residues G140-G149 in drug action and resistance.
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probing hiv 1 Integrase inhibitor binding sites with position specific Integrase dna cross linking assays
Molecular Pharmacology, 2007Co-Authors: Allison A Johnson, Christophe Marchand, Sachindra S Patil, Roberta Costi, Roberto Di Santo, Terrence R Burke, Yves PommierAbstract:HIV-1 Integrase binds site-specifically to the ends of the viral cDNA. We used two HIV-1 Integrase-DNA cross-linking assays to probe the binding sites of Integrase inhibitors from different chemical families and with different strand transfer selectivities. The disulfide assay probes cross-linking between the Integrase residue 148 and the 5'-terminal cytosine of the viral cDNA, and the Schiff base assay probes cross-linking between an Integrase lysine residue and an abasic site placed at selected positions in the viral cDNA. Cross-linking interference by eight Integrase inhibitors shows that the most potent cross-linking inhibitors are 3'-processing inhibitors, indicating that cross-linking assays probe the donor viral cDNA (donor binding site). In contrast, strand transfer-selective inhibitors provide weak cross-linking interference, consistent with their binding to a specific acceptor (cellular DNA) site. Docking and crystal structure studies illustrate specific Integrase-inhibitor contacts that prevent cross-linking formation. Four inhibitors that prevented Schiff base cross-linking to the conserved 3'-terminal adenine position were examined for inhibition at various positions within the terminal 21 bases of the viral cDNA. Two of them selectively inhibited upper strand cross-linking, whereas the other two had a more global effect on Integrase-DNA binding. These findings have implications for elucidating inhibitor binding sites and mechanisms of action. The cross-linking assays also provide clues to the molecular interactions between Integrase and the viral cDNA.
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Integrase inhibitors to treat hiv aids
Nature Reviews Drug Discovery, 2005Co-Authors: Yves Pommier, Allison A Johnson, Christophe MarchandAbstract:HIV Integrase is a rational target for treating HIV infection and preventing AIDS. It took approximately 12 years to develop clinically usable inhibitors of Integrase, and Phase I clinical trials of Integrase inhibitors have just begun. This review focuses on the molecular basis and rationale for developing Integrase inhibitors. The main classes of lead compounds are also described, as well as the concept of interfacial inhibitors of protein-nucleic-acid interactions that might apply to the clinically used strand-transfer inhibitors.
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effect of hiv Integrase inhibitors on the rag1 2 recombinase
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Meni Melek, Nouri Neamati, Terrence R Burke, Yves Pommier, Jessica M Jones, Mary H Odea, Godwin Pais, Martin GellertAbstract:Assembly of functional Ig and T cell receptor genes by V(D)J recombination depends on site-specific cleavage of chromosomal DNA by the RAG1/2 recombinase. As RAG1/2 action has mechanistic similarities to DNA transposases and Integrases such as HIV-1 Integrase, we sought to determine how Integrase inhibitors of the diketo acid type would affect the various activities of RAG1/2. Both of the inhibitors we tested interfered with DNA cleavage and disintegration activities of RAG1/2, apparently by disrupting interaction with the DNA motifs bound specifically by the recombinase. The inhibitors did not ablate RAG1/2's transposition activity or capture of nonspecific transpositional target DNA, suggesting this DNA occupies a site on the recombinase different from that used for specific binding. These results further underscore the similarities between RAG1/2 and Integrase and suggest that certain Integrase inhibitors may have the potential to interfere with aspects of B and T cell development.
Alan Engelman - One of the best experts on this subject based on the ideXlab platform.
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close up hiv siv intasome structures shed new light on Integrase inhibitor binding and viral escape mechanisms
FEBS Journal, 2021Co-Authors: Alan Engelman, Peter CherepanovAbstract:Integrase strand transfer inhibitors (INSTIs) are important components of drug formulations that are used to treat people living with HIV, and second-generation INSTIs dolutegravir and bictegravir impart high barriers to the development of drug resistance. Reported 10 years ago, X-ray crystal structures of prototype foamy virus (PFV) intasome complexes explained how INSTIs bind Integrase to inhibit strand transfer activity and provided initial glimpses into mechanisms of drug resistance. However, comparatively low sequence identity between PFV and HIV-1 Integrases limited the depth of information that could be gleaned from the surrogate model system. Recent high-resolution structures of HIV-1 intasomes as well as intasomes from a closely related strain of simian immunodeficiency virus (SIV), which were determined using single-particle cryogenic electron microscopy, have overcome this limitation. The new structures reveal the binding modes of several advanced INSTI compounds to the HIV/SIV Integrase active site and critically inform the structural basis of drug resistance. These findings will help guide the continued development of this important class of antiretroviral therapeutics.
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Integrase rna interactions underscore the critical role of Integrase in hiv 1 virion morphogenesis
eLife, 2020Co-Authors: Jennifer L Elliott, Alan Engelman, Pratibha C Koneru, Jenna E Eschbach, Maritza Puraychavez, Dana Townsend, Dana Q Lawson, Mamuka Kvaratskhelia, Sebla B KutluayAbstract:A large number of human immunodeficiency virus 1 (HIV-1) Integrase (IN) alterations, referred to as class II substitutions, exhibit pleiotropic effects during virus replication. However, the underlying mechanism for the class II phenotype is not known. Here we demonstrate that all tested class II IN substitutions compromised IN-RNA binding in virions by one of the three distinct mechanisms: (i) markedly reducing IN levels thus precluding the formation of IN complexes with viral RNA; (ii) adversely affecting functional IN multimerization and consequently impairing IN binding to viral RNA; and (iii) directly compromising IN-RNA interactions without substantially affecting IN levels or functional IN multimerization. Inhibition of IN-RNA interactions resulted in the mislocalization of viral ribonucleoprotein complexes outside the capsid lattice, which led to premature degradation of the viral genome and IN in target cells. Collectively, our studies uncover causal mechanisms for the class II phenotype and highlight an essential role of IN-RNA interactions for accurate virion maturation.
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Integrase rna interactions underscore the critical role of Integrase in hiv 1 virion morphogenesis
bioRxiv, 2019Co-Authors: Jennifer L Elliott, Alan Engelman, Pratibha C Koneru, Jenna E Eschbach, Dana Townsend, Dana Q Lawson, Mamuka Kvaratskhelia, Maritza Puray Chavez, Sebla B KutluayAbstract:ABSTRACT A large number of HIV-1 Integrase (IN) alterations, referred to as class II substitutions, exhibit pleotropic effects during virus replication. However, the underlying mechanism for the class II phenotype is not known. Here we demonstrate that all tested class II IN substitutions compromised IN-RNA binding in virions by one of three distinct mechanisms: i) markedly reducing IN levels thus precluding formation of IN complexes with viral RNA; ii) adversely affecting functional IN multimerization and consequently impairing IN binding to viral RNA; iii) directly compromising IN-RNA interactions without substantially affecting IN levels or functional IN multimerization. Inhibition of IN-RNA interactions resulted in mislocalization of the viral ribonucleoprotein complexes outside the capsid lattice, which led to premature degradation of the viral genome and IN in target cells. Collectively, our studies uncover causal mechanisms for the class II phenotype and highlight an essential role of IN-RNA interactions for accurate virion maturation.
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hiv 1 Integrase tetramers are the antiviral target of pyridine based allosteric Integrase inhibitors
eLife, 2019Co-Authors: Pratibha C Koneru, Ashwanth C Francis, Nanjie Deng, Stephanie V Rebensburg, Ashley C Hoyte, Jared J Lindenberger, Daniel Aduampratwum, Ross C Larue, Michael F Wempe, Alan EngelmanAbstract:Allosteric HIV-1 Integrase (IN) inhibitors (ALLINIs) are a promising new class of antiretroviral agents that disrupt proper viral maturation by inducing hyper-multimerization of IN. Here we show that lead pyridine-based ALLINI KF116 exhibits striking selectivity for IN tetramers versus lower order protein oligomers. IN structural features that are essential for its functional tetramerization and HIV-1 replication are also critically important for KF116 mediated higher-order IN multimerization. Live cell imaging of single viral particles revealed that KF116 treatment during virion production compromises the tight association of IN with capsid cores during subsequent infection of target cells. We have synthesized the highly active (-)-KF116 enantiomer, which displayed EC50 of ~7 nM against wild type HIV-1 and ~10 fold higher, sub-nM activity against a clinically relevant dolutegravir resistant mutant virus suggesting potential clinical benefits for complementing dolutegravir therapy with pyridine-based ALLINIs.
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Retroviral Integrase Proteins and HIV-1 DNA Integration
The Journal of biological chemistry, 2012Co-Authors: Lavanya Krishnan, Alan EngelmanAbstract:Retroviral Integrases catalyze two reactions, 3′-processing of viral DNA ends, followed by integration of the processed ends into chromosomal DNA. X-ray crystal structures of Integrase-DNA complexes from prototype foamy virus, a member of the Spumavirus genus of Retroviridae, have revealed the structural basis of integration and how clinically relevant Integrase strand transfer inhibitors work. Underscoring the translational potential of targeting virus-host interactions, small molecules that bind at the host factor lens epithelium-derived growth factor/p75-binding site on HIV-1 Integrase promote dimerization and inhibit Integrase-viral DNA assembly and catalysis. Here, we review recent advances in our knowledge of HIV-1 DNA integration, as well as future research directions.
Frederic D Bushman - One of the best experts on this subject based on the ideXlab platform.
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bromodomain and extra terminal bet proteins target moloney murine leukemia virus integration to transcription start sites
Retrovirology, 2013Co-Authors: Jan De Riick, Christine De Kogel, Jonas Demeulemeester, Sofie Vets, Nirav Malani, Frederic D Bushman, Katrien Busschots, Steven Husson, Rik Gijsbers, Zeger DebyserAbstract:A hallmark of retroviral replication is stable integration of the viral genome in the host cell DNA. This characteristic makes retroviral-derived vector particles attractive vehicles for gene therapy. However, retroviral integration is not a random process. Lentiviruses preferentially integrate in the body of active transcription units, while gammaretroviruses, including Moloney Murine Leukemia Virus (MLV), favour transcription start sites and CpG islands. In clinical trials using gammaretroviral vectors for gene therapy, leukemogenesis has been associated with integration of vectors near oncogene transcription start sites. We found that the bromodomain and extra-terminal (BET) proteins (BRD2, BRD3 and BRD4) interact with MLV Integrase and direct integration towards transcription start regions. BET proteins specifically bind and co-localize with the gammaretrovirus Integrase protein in the nucleus of the cell. The interaction is gammaretroviral-specific and mediated by the Integrase C-terminal domain and the BET extraterminal (ET) domain as determined by co-immunoprecipitation assays and in an Alphascreen assay using recombinant proteins. Interfering with chromatin interaction of BET proteins via specific bromodomain inhibitors JQ1 and l-BET decreases MLV virus replication and MLV vector transduction 5-to 10-fold, while HIV vector transduction is not affected. Analysis of viral DNA intermediates by quantitative PCR revealed a block at the integration step. In addition, bromodomain inhibitors do not have an effect on the late steps of viral replication. MLV integration site distribution analysis revealed a strong correlation with the BET protein chromatin binding profile. Finally, expression of an artificial fusion protein that merges the BET Integrase binding domain with the chromatin interaction domain of the lentiviral targeting factor LEDGF/p75, retargets MLV integration into the body of actively transcribed genes, paralleling the Human Immunodeficiency Virus (HIV) integration pattern. Our results explain the molecular mechanism behind gammaretroviral integration site targeting and suggest methods for engineering gammaretroviral vectors with a safer integration site profile.
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rapid microtiter assays for poxvirus topoisomerase mammalian type ib topoisomerase and hiv 1 Integrase application to inhibitor isolation
Nucleic Acids Research, 2000Co-Authors: Young Hwang, Denise Rhodes, Frederic D BushmanAbstract:We have developed microtiter assays for detecting catalysis by type IB topoisomerases and retroviral Integrases. Each assay employs model DNA substrates containing biotin in one strand and digoxigenin in another. In each case action of the enzyme results in the formation of a single DNA strand containing both groups. This allows the reaction product to be quantified by capturing biotinylated product DNA on avidin-coated plates followed by detection using an anti-digoxigenin ELISA. The order of addition of reactants and inhibitors can be varied to distinguish effects of test compounds on different steps in the reaction. These assays were used to screen compound libraries for inhibitors active against mammalian topoisomerase or HIV Integrase. We identified (–)-epigallocatechin 3-O-gallate, as a potent inhibitor of religation by mammalian topoisomerase (IC 50 of 26 nM), potentially explaining the anti-cancer properties previously attributed to this compound. New Integrase inhibitors were also identified. A similar strategy may be used to develop microtiter assays for many further DNA modifying enzymes.
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human immunodeficiency virus type 1 preintegration complexes studies of organization and composition
Journal of Virology, 1997Co-Authors: Michael D Miller, Chris M Farnet, Frederic D BushmanAbstract:We have investigated the organization and function of human immunodeficiency virus type 1 (HIV-1) preintegration complexes (PICs), the large nucleoprotein particles that carry out cDNA integration in vivo. PICs can be isolated from HIV-1-infected cells, and such particles are capable of carrying out integration reactions in vitro. We find that although the PICs are large, the cDNA must be condensed to fit into the measured volume. The ends of the cDNA are probably linked by a protein bridge, since coordinated joining of the two ends is not disrupted by cleaving the cDNA internally with a restriction enzyme. cDNA ends in PICs were protected from digestion by added exonucleases, probably due to binding of proteins. The intervening cDNA, in contrast, was susceptible to attack by endonucleases. Previous work has established that the virus-encoded Integrase protein is present in PICs, and we have reported recently that the host protein HMG I(Y) is also present. Here we report that the viral matrix and reverse transcriptase (RT) proteins also cofractionated with PICs through several steps whereas capsid and nucleocapsid proteins dissociated. These data support a model of PIC organization in which the cDNA is condensed in a partially disassembled remnant of the viral core, with proteins tightly associated at the apposed cDNA ends but loosely associated with the intervening cDNA. In characterizing the structure of the cDNA ends, we found that the U5 DNA ends created by RT were ragged, probably due to the terminal transferase activity of RT. Only molecules correctly cleaved by Integrase protein at the 3* ends were competent to integrate, suggesting that one role for terminal cleavage by Integrase may be to create a defined end at otherwise heterogeneous cDNA termini.
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domains of the Integrase protein of human immunodeficiency virus type 1 responsible for polynucleotidyl transfer and zinc binding
Proceedings of the National Academy of Sciences of the United States of America, 1993Co-Authors: Frederic D Bushman, Alan Engelman, Ira Palmer, Paul T Wingfield, Robert CraigieAbstract:Abstract The Integrase protein of human immunodeficiency virus type 1 carries out a set of polynucleotidyl transfer reactions that result in the covalent attachment of the retroviral cDNA to host DNA. We have analyzed the activities of a set of deletion derivatives of the Integrase protein. The analysis reveals that a central domain of only 137 amino acids is sufficient in vitro to catalyze a subset of the reactions carried out by the complete protein. This polypeptide contains an amino acid sequence motif, Asp-Xaa39-58-Asp-Xaa35-Glu (DX39-58DX35E, where X and the subscript indicate the intervening amino acids between the invariant acidic residues), that is found in the Integrases of retroviruses and retrotransposons and also the transposase proteins of some bacterial transposable elements. We also find that the Integrase protein can bind Zn2+, and the histidine and cysteine residues of another conserved motif (HX3-7HX23-32CX2C) are required for efficient Zn2+ binding. The activities displayed by deletion mutants suggest to us possible functions for the various parts of Integrase.
Ulrich Dobrindt - One of the best experts on this subject based on the ideXlab platform.
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sub inhibitory concentrations of sos response inducing antibiotics stimulate Integrase expression and excision of pathogenicity islands in uropathogenic escherichia coli strain 536
International Journal of Medical Microbiology, 2020Co-Authors: Marco Chitto, Michael Berger, Luisa Klotz, Ulrich DobrindtAbstract:Abstract Urinary tract infections are one of the most common bacterial infections and a major public health problem. The predominant causative agents are uropathogenic Escherichia coli. These strains differ from commensal E. coli by the presence of additional horizontally acquired chromosomal material, so-called pathogenicity islands, which encode traits that promote efficient bacterial colonization of the urinary tract. Uropathogenic model strain E. coli 536 possesses six archetypal pathogenicity islands. Bacteriophage-like Integrases encoded by each pathogenicity island contribute to island instability. To learn more about the stability of these six islands and factors controlling their stability we constructed two chromosomal reporter systems for the measurement of island loss, as well as for the measurement of the promoter activity of the six island-associated Integrase genes at the population level. We used these reporter gene modules to analyze the role of SOS response in island instability. Tests with subinhibitory concentrations of different antibiotics, including many drugs commonly used for the treatment of urinary tract infection, indicated that only SOS response-inducing antibiotics led to an increased loss of islands which was always associated with an increase in the bacterial subpopulations showing high Integrase promoter activity. This suggests that island excision correlates with the expression of the cognate Integrase. Our reporter modules are valuable tools to investigate the impact of various growth conditions on genome plasticity. Furthermore, a better understanding of the conditions, which affect bacterial Integrase expression may open ways to specifically manipulate the genome content of bacterial pathogens by increasing pathogenicity island deletion rates in infecting or colonizing bacteria, thus leading to the attenuation of bacterial pathogens.
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delineation of the recombination sites necessary for integration of pathogenicity islands ii and iii into the escherichia coli 536 chromosome
Molecular Microbiology, 2008Co-Authors: Ulrich Dobrindt, Bianca Hochhut, Barbara Middendorf, Caroline Wilde, Didier Mazel, Frederique Le Roux, Elisabeth Carniel, Jorg HackerAbstract:In uropathogenic Escherichia coil strain 536, six pathogenicity islands (PAls) encode key virulence factors. All PAls except PAI IV 536 are flanked by direct repeats and four of them encode Integrases responsible for their chromosomal excision. To study recombination sites used for the integration by PAI II 536 and III 536 Integrases, we measured site-specific recombination between the chromosomal integration site attB, and the PAI-specific attachment site attP. We show that PAI III 536 IntB, but not IntA, mediates PAI III 536 integration. Studies of integrative recombination sites of both PAls show that, when using a large cognate attP site (839 bp for PAI II 536 and 268 bp for PAI III 536 ), PAI II 536 and III 536 attB sites could be reduced to 16 bp and 20 bp, respectively, without affecting recombination. Further reduction to 14 bp for PAI II 536 and 13 bp for PAI III 536 diminished recombination efficiency. Surprisingly, attP sites could also be reduced to 14 bp (PAI II 536 ) and 20 bp (PAI III 536 ). The integration host factor (IHF) and the DNA-bending HU protein do not influence PAI II 536 recombination, but IHF enhances PAI-III 536 excision and negatively affects its integration. These data suggest that PAI intasomes differ from those of lambda and P4 Integrase paradigms.
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role of pathogenicity island associated Integrases in the genome plasticity of uropathogenic escherichia coli strain 536
Molecular Microbiology, 2006Co-Authors: Bianca Hochhut, Ulrich Dobrindt, Barbara Middendorf, Caroline Wilde, Elisabeth Carniel, Gudrun Balling, Elzbieta Brzuszkiewicz, Gerhard Gottschalk, Jorg HackerAbstract:The genome of uropathogenic Escherichia coli isolate 536 contains five well-characterized pathogenicity islands (PAIs) encoding key virulence factors of this strain. Except PAI IV(536), the four other PAIs of strain 536 are flanked by direct repeats (DRs), carry intact Integrase genes and are able to excise site-specifically from the chromosome. Genome screening of strain 536 identified a sixth putative asnW-associated PAI. Despite the presence of DRs and an intact Integrase gene, excision of this island was not detected. To investigate the role of PAI-encoded Integrases for the recombination process the int genes of each unstable island of strain 536 were inactivated. For PAI I(536) and PAI II(536), their respective P4-like Integrase was required for their excision. PAI III(536) carries two Integrase genes, intA, encoding an SfX-like Integrase, and intB, coding for an Integrase with weak similarity to P4-like Integrases. Only intB was required for site-specific excision of this island. For PAI V(536), excision could not be abolished after deleting its P4-like Integrase gene but additional deletion of the PAI II(536)-specific Integrase gene was required. Therefore, although all mediated by P4-like Integrases, the activity of the PAI excision machinery is most often restricted to its cognate island. This work also demonstrates for the first time the existence of a cross-talk between Integrases of different PAIs and shows that this cross-talk is unidirectional.
Jorg Hacker - One of the best experts on this subject based on the ideXlab platform.
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delineation of the recombination sites necessary for integration of pathogenicity islands ii and iii into the escherichia coli 536 chromosome
Molecular Microbiology, 2008Co-Authors: Ulrich Dobrindt, Bianca Hochhut, Barbara Middendorf, Caroline Wilde, Didier Mazel, Frederique Le Roux, Elisabeth Carniel, Jorg HackerAbstract:In uropathogenic Escherichia coil strain 536, six pathogenicity islands (PAls) encode key virulence factors. All PAls except PAI IV 536 are flanked by direct repeats and four of them encode Integrases responsible for their chromosomal excision. To study recombination sites used for the integration by PAI II 536 and III 536 Integrases, we measured site-specific recombination between the chromosomal integration site attB, and the PAI-specific attachment site attP. We show that PAI III 536 IntB, but not IntA, mediates PAI III 536 integration. Studies of integrative recombination sites of both PAls show that, when using a large cognate attP site (839 bp for PAI II 536 and 268 bp for PAI III 536 ), PAI II 536 and III 536 attB sites could be reduced to 16 bp and 20 bp, respectively, without affecting recombination. Further reduction to 14 bp for PAI II 536 and 13 bp for PAI III 536 diminished recombination efficiency. Surprisingly, attP sites could also be reduced to 14 bp (PAI II 536 ) and 20 bp (PAI III 536 ). The integration host factor (IHF) and the DNA-bending HU protein do not influence PAI II 536 recombination, but IHF enhances PAI-III 536 excision and negatively affects its integration. These data suggest that PAI intasomes differ from those of lambda and P4 Integrase paradigms.
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role of pathogenicity island associated Integrases in the genome plasticity of uropathogenic escherichia coli strain 536
Molecular Microbiology, 2006Co-Authors: Bianca Hochhut, Ulrich Dobrindt, Barbara Middendorf, Caroline Wilde, Elisabeth Carniel, Gudrun Balling, Elzbieta Brzuszkiewicz, Gerhard Gottschalk, Jorg HackerAbstract:The genome of uropathogenic Escherichia coli isolate 536 contains five well-characterized pathogenicity islands (PAIs) encoding key virulence factors of this strain. Except PAI IV(536), the four other PAIs of strain 536 are flanked by direct repeats (DRs), carry intact Integrase genes and are able to excise site-specifically from the chromosome. Genome screening of strain 536 identified a sixth putative asnW-associated PAI. Despite the presence of DRs and an intact Integrase gene, excision of this island was not detected. To investigate the role of PAI-encoded Integrases for the recombination process the int genes of each unstable island of strain 536 were inactivated. For PAI I(536) and PAI II(536), their respective P4-like Integrase was required for their excision. PAI III(536) carries two Integrase genes, intA, encoding an SfX-like Integrase, and intB, coding for an Integrase with weak similarity to P4-like Integrases. Only intB was required for site-specific excision of this island. For PAI V(536), excision could not be abolished after deleting its P4-like Integrase gene but additional deletion of the PAI II(536)-specific Integrase gene was required. Therefore, although all mediated by P4-like Integrases, the activity of the PAI excision machinery is most often restricted to its cognate island. This work also demonstrates for the first time the existence of a cross-talk between Integrases of different PAIs and shows that this cross-talk is unidirectional.
