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Alan Engelman - One of the best experts on this subject based on the ideXlab platform.

  • retroviral DNA Integration
    Chemical Reviews, 2016
    Co-Authors: Paul Lesbats, Alan Engelman, Peter Cherepanov
    Abstract:

    The Integration of a DNA copy of the viral RNA genome into host chromatin is the defining step of retroviral replication. This enzymatic process is catalyzed by the virus-encoded integrase protein, which is conserved among retroviruses and LTR-retrotransposons. Retroviral Integration proceeds via two integrase activities: 3′-processing of the viral DNA ends, followed by the strand transfer of the processed ends into host cell chromosomal DNA. Herein we review the molecular mechanism of retroviral DNA Integration, with an emphasis on reaction chemistries and architectures of the nucleoprotein complexes involved. We additionally discuss the latest advances on anti-integrase drug development for the treatment of AIDS and the utility of integrating retroviral vectors in gene therapy applications.

  • sites of retroviral DNA Integration from basic research to clinical applications
    Critical Reviews in Biochemistry and Molecular Biology, 2016
    Co-Authors: Erik Serrao, Alan Engelman
    Abstract:

    One of the most crucial steps in the life cycle of a retrovirus is the Integration of the viral DNA (vDNA) copy of the RNA genome into the genome of an infected host cell. Integration provides for efficient viral gene expression as well as for the segregation of viral genomes to daughter cells upon cell division. Some integrated viruses are not well expressed, and cells latently infected with human immunodeficiency virus type 1 (HIV-1) can resist the action of potent antiretroviral drugs and remain dormant for decades. Intensive research has been dedicated to understanding the catalytic mechanism of Integration, as well as the viral and cellular determinants that influence Integration site distribution throughout the host genome. In this review, we summarize the evolution of techniques that have been used to recover and map retroviral Integration sites, from the early days that first indicated that Integration could occur in multiple cellular DNA locations, to current technologies that map upwards of millions of unique Integration sites from single in vitro Integration reactions or cell culture infections. We further review important insights gained from the use of such mapping techniques, including the monitoring of cell clonal expansion in patients treated with retrovirus-based gene therapy vectors, or patients with acquired immune deficiency syndrome (AIDS) on suppressive antiretroviral therapy (ART). These insights span from integrase (IN) enzyme sequence preferences within target DNA (tDNA) at the sites of Integration, to the roles of host cellular proteins in mediating global Integration distribution, to the potential relationship between genomic location of vDNA Integration site and retroviral latency.

  • engineered hyperactive integrase for concerted hiv 1 DNA Integration
    PLOS ONE, 2014
    Co-Authors: Kellie A Jurado, Alan Engelman, Shiqiang Lin, Robert Craigie
    Abstract:

    The DNA cutting and joining reactions of HIV-1 Integration are catalyzed by integrase (IN), a viral protein that functions as a tetramer bridging the two viral DNA ends (intasome). Two major obstacles for biochemical and structural studies of HIV-1 intasomes are 1) the low efficiency of assembly with oligonucleotide DNA substrates, and 2) the non-specific aggregation of both intasomes and free IN in the reaction mixture. By fusing IN with a small non-specific DNA binding protein, Sulfolobus solfataricus chromosomal protein Sso7d (PDB: 1BNZ), we have engineered a highly soluble and hyperactive IN. Unlike wild-type IN, it efficiently catalyzes intasome assembly and concerted Integration with oligonucleotide DNA substrates. The fusion IN protein also functions to integrate viral reverse transcripts during HIV-infection. The hyperactive HIV-1 IN may assist in facilitating future biochemical and structural studies of HIV-1 intasomes. Understanding the mechanistic basis of the Sso7d-IN fusion protein could provide insight into the factors that have hindered biophysical studies of wild-type HIV-1 IN and intasomes.

  • differential effects of human immunodeficiency virus type 1 capsid and cellular factors nucleoporin 153 and ledgf p75 on the efficiency and specificity of viral DNA Integration
    Journal of Virology, 2013
    Co-Authors: Yasuhiro Koh, Andrea L. Ferris, Steven J. Smith, Kenneth A Matreyek, Kyeongeun Lee, Vineet N Kewalramani, Stephen H Hughes, Alan Engelman
    Abstract:

    Retroviruses integrate into cellular DNA nonrandomly. Lentiviruses such as human immunodeficiency virus type 1 (HIV-1) favor the bodies of active genes and gene-enriched transcriptionally active regions of chromosomes. The interaction between lentiviral integrase and the cellular protein lens epithelium-derived growth factor (LEDGF)/p75 underlies the targeting of gene bodies, whereas recent research has highlighted roles for the HIV-1 capsid (CA) protein and cellular factors implicated in viral nuclear import, including transportin 3 (TNPO3) and nucleoporin 358 (NUP358), in the targeting of gene-dense regions of chromosomes. Here, we show that CA mutations, which include the substitution of Asp for Asn74 (N74D), significantly reduce the dependency of HIV-1 on LEDGF/p75 during infection and that this difference correlates with the efficiency of viral DNA Integration. The distribution of Integration sites mapped by Illumina sequencing confirms that the N74D mutation reduces Integration into gene-rich regions of chromosomes and gene bodies and reveals previously unrecognized roles for NUP153 (another HIV-1 cofactor implicated in viral nuclear import) and LEDGF/p75 in the targeting of the viral preIntegration complex to gene-dense regions of chromatin. A role for the CA protein in determining the dependency of HIV-1 on LEDGF/p75 during infection highlights a connection between the viral capsid and chromosomal DNA Integration.

  • Retroviral Integrase Proteins and HIV-1 DNA Integration
    The Journal of biological chemistry, 2012
    Co-Authors: Lavanya Krishnan, Alan Engelman
    Abstract:

    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.

Robert Craigie - One of the best experts on this subject based on the ideXlab platform.

  • human three prime repair exonuclease 1 promotes hiv 1 Integration by preferentially degrading unprocessed viral DNA
    Journal of Virology, 2021
    Co-Authors: Benemorom Davids, Robert Craigie, Muthukumar Balasubramaniam, Nicklas Sapp, Prem Prakash, Shalonda M Ingram, Thomas Hollis, Jui Pandhare, Chandravanu Dash
    Abstract:

    Three prime repair exonuclease 1 (TREX1) is the most abundant 3'→5' exonuclease in mammalian cells. It has been suggested that TREX1 degrades HIV-1 DNA to enable the virus to evade the innate immune system. However, the exact role of TREX1 during early steps of HIV-1 infection is not clearly understood. In this study, we report that HIV-1 infection is associated with upregulation, perinuclear accumulation, and nuclear localization of TREX1. However, TREX1 overexpression did not affect reverse transcription or nuclear entry of the virus. Surprisingly, HIV-1 DNA Integration was increased in TREX1-overexpressing cells, suggesting a role of the exonuclease in the post-nuclear entry step of infection. Accordingly, preIntegration complexes (PICs) extracted from TREX1-overexpressing cells retained higher levels of DNA Integration activity. TREX1 depletion resulted in reduced levels of proviral Integration, and PICs formed in TREX1-depleted cells retained lower DNA Integration activity. Addition of purified TREX1 to PICs also enhanced DNA Integration activity, suggesting that TREX1 promotes HIV-1 Integration by stimulating PIC activity. To understand the mechanism, we measured TREX1 exonuclease activity on substrates containing viral DNA ends. These studies revealed that TREX1 preferentially degrades the unprocessed viral DNA, but the Integration-competent 3'-processed viral DNA remains resistant to degradation. Finally, we observed that TREX1 addition stimulates the activity of HIV-1 intasomes assembled with the unprocessed viral DNA but not that of intasomes containing the 3'-processed viral DNA. These biochemical analyses provide a mechanism by which TREX1 directly promotes HIV-1 Integration. Collectively, our study demonstrates that HIV-1 infection upregulates TREX1 to facilitate viral DNA Integration. IMPORTANCE Productive HIV-1 infection is dependent on a number of cellular factors. Therefore, a clear understanding of how the virus exploits the cellular machinery will identify new targets for inhibiting HIV-1 infection. The three prime repair exonuclease 1 (TREX1) is the most active cellular exonuclease in mammalian cells. It has been reported that TREX1 prevents accumulation of HIV-1 DNA and enables the virus to evade the host innate immune response. Here, we show that HIV-1 infection results in the upregulation, perinuclear accumulation, and nuclear localization of TREX1. We also provide evidence that TREX1 promotes HIV-1 Integration by preferentially degrading viral DNAs that are incompatible with chromosomal insertion. These observations identify a novel role of TREX1 in a post-nuclear entry step of HIV-1 infection.

  • engineered hyperactive integrase for concerted hiv 1 DNA Integration
    PLOS ONE, 2014
    Co-Authors: Kellie A Jurado, Alan Engelman, Shiqiang Lin, Robert Craigie
    Abstract:

    The DNA cutting and joining reactions of HIV-1 Integration are catalyzed by integrase (IN), a viral protein that functions as a tetramer bridging the two viral DNA ends (intasome). Two major obstacles for biochemical and structural studies of HIV-1 intasomes are 1) the low efficiency of assembly with oligonucleotide DNA substrates, and 2) the non-specific aggregation of both intasomes and free IN in the reaction mixture. By fusing IN with a small non-specific DNA binding protein, Sulfolobus solfataricus chromosomal protein Sso7d (PDB: 1BNZ), we have engineered a highly soluble and hyperactive IN. Unlike wild-type IN, it efficiently catalyzes intasome assembly and concerted Integration with oligonucleotide DNA substrates. The fusion IN protein also functions to integrate viral reverse transcripts during HIV-infection. The hyperactive HIV-1 IN may assist in facilitating future biochemical and structural studies of HIV-1 intasomes. Understanding the mechanistic basis of the Sso7d-IN fusion protein could provide insight into the factors that have hindered biophysical studies of wild-type HIV-1 IN and intasomes.

  • hiv DNA Integration
    Cold Spring Harbor Perspectives in Medicine, 2012
    Co-Authors: Robert Craigie, Frederic D Bushman
    Abstract:

    Retroviruses are distinguished from other viruses by two characteristic steps in the viral replication cycle. The first is reverse transcription, which results in the production of a double-stranded DNA copy of the viral RNA genome, and the second is Integration, which results in covalent attachment of the DNA copy to host cell DNA. The initial catalytic steps of the Integration reaction are performed by the virus-encoded integrase (IN) protein. The chemistry of the IN-mediated DNA breaking and joining steps is well worked out, and structures of IN-DNA complexes have now clarified how the overall complex assembles. Methods developed during these studies were adapted for identification of IN inhibitors, which received FDA approval for use in patients in 2007. At the chromosomal level, HIV Integration is strongly favored in active transcription units, which may promote efficient viral gene expression after Integration. HIV IN binds to the cellular factor LEDGF/p75, which promotes efficient infection and tethers IN to favored target sites. The HIV Integration machinery must also interact with many additional host factors during infection, including nuclear trafficking and pore proteins during nuclear entry, histones during initial target capture, and DNA repair proteins during completion of the DNA joining steps. Models for some of the molecular mechanisms involved have been proposed, but important details remain to be clarified.

  • retroviral DNA Integration reaction pathway and critical intermediates
    The EMBO Journal, 2006
    Co-Authors: Michiyo Mizuuchi, Terrence R Burke, Robert Craigie
    Abstract:

    The key DNA cutting and joining steps of retroviral DNA Integration are carried out by the viral integrase protein. Structures of the individual domains of integrase have been determined, but their organization in the active complex with viral DNA is unknown. We show that HIV-1 integrase forms stable synaptic complexes in which a tetramer of integrase is stably associated with a pair of viral DNA ends. The viral DNA is processed within these complexes, which go on to capture the target DNA and integrate the viral DNA ends. The joining of the two viral DNA ends to target DNA occurs sequentially, with a stable intermediate complex in which only one DNA end is joined. The Integration product also remains stably associated with integrase and likely requires disassembly before completion of the Integration process by cellular enzymes. The results define the series of stable nucleoprotein complexes that mediate retroviral DNA Integration.

  • hiv 1 DNA Integration mechanism of viral DNA cleavage and DNA strand transfer
    Cell, 1991
    Co-Authors: Alan Engelman, Kiyoshi Mizuuchi, Robert Craigie
    Abstract:

    Retroviral DNA Integration involves a coordinated set of DNA cutting and joining reactions. Linear viral DNA is cleaved at each 3' end to generate the precursor ends for Integration. The resulting recessed 3' ends are inserted into target DNA by a subsequent DNA strand transfer reaction. Purified HIV-1 Integration protein carries out both of these steps in vitro. Two novel forms of the dinucleotide cleaved from HIV-1 DNA were identified and one, a cyclic dinucleotide, was used to analyze the stereochemical course of viral DNA cleavage. Both viral DNA cleavage and DNA strand transfer display inversion at chiral phosphorothioates during the course of the reaction. These results suggest that both reactions occur by a one-step mechanism without involvement of a covalent protein-DNA intermediate.

Peter Cherepanov - One of the best experts on this subject based on the ideXlab platform.

  • retroviral DNA Integration
    Chemical Reviews, 2016
    Co-Authors: Paul Lesbats, Alan Engelman, Peter Cherepanov
    Abstract:

    The Integration of a DNA copy of the viral RNA genome into host chromatin is the defining step of retroviral replication. This enzymatic process is catalyzed by the virus-encoded integrase protein, which is conserved among retroviruses and LTR-retrotransposons. Retroviral Integration proceeds via two integrase activities: 3′-processing of the viral DNA ends, followed by the strand transfer of the processed ends into host cell chromosomal DNA. Herein we review the molecular mechanism of retroviral DNA Integration, with an emphasis on reaction chemistries and architectures of the nucleoprotein complexes involved. We additionally discuss the latest advances on anti-integrase drug development for the treatment of AIDS and the utility of integrating retroviral vectors in gene therapy applications.

  • structural insights into the retroviral DNA Integration apparatus
    Current Opinion in Structural Biology, 2011
    Co-Authors: Peter Cherepanov, Goedele N Maertens, Stephen Hare
    Abstract:

    Retroviral replication depends on successful Integration of the viral genetic material into a host cell chromosome. Virally encoded integrase, an enzyme from the DDE(D) nucleotidyltransferase superfamily, is responsible for the key DNA cutting and joining steps associated with this process. Insights into the structural and mechanistic aspects of Integration are directly relevant for the development of antiretroviral drugs. Recent breakthroughs have led to biochemical and structural characterization of the principal Integration intermediates revealing the tetramer of integrase that catalyzes insertion of both 3′ viral DNA ends into a sharply bent target DNA. This review discusses the mechanism of retroviral DNA Integration and the mode of action of HIV-1 integrase strand transfer inhibitors in light of the recent visualization of the prototype foamy virus intasome, target DNA capture and strand transfer complexes.

  • structural biology of retroviral DNA Integration
    Virology, 2011
    Co-Authors: Lavanya Krishnan, Peter Cherepanov, Alan Engelman
    Abstract:

    Three-dimensional macromolecular structures shed critical light on biological mechanism and facilitate development of small molecule inhibitors. Clinical success of raltegravir, a potent inhibitor of HIV-1 integrase, demonstrated the utility of this viral DNA recombinase as an antiviral target. A variety of partial integrase structures reported in the past 16 years have been instrumental and very informative to the field. Nonetheless, because integrase protein fragments are unable to functionally engage the viral DNA substrate critical for strand transfer inhibitor binding, the early structures did little to materially impact drug development efforts. However, recent results based on prototype foamy virus integrase have fully reversed this trend, as a number of X-ray crystal structures of active integrase-DNA complexes revealed key mechanistic details and moreover established the foundation of HIV-1 integrase strand transfer inhibitor action. In this review we discuss the landmarks in the progress of integrase structural biology during the past 17 years.

Myeongje Cho - One of the best experts on this subject based on the ideXlab platform.

  • High aspect ratio nanomaterials enable delivery of functional genetic material without DNA Integration in mature plants
    Nature Nanotechnology, 2019
    Co-Authors: Gozde S Demirer, Huan Zhang, Juliana L Matos, Roger Chang, Linda Chio, Natalie S. Goh, Abhishek J Aditham, Francis J. Cunningham, Younghun Sung, Myeongje Cho
    Abstract:

    Genetic engineering of plants is at the core of sustainability efforts, natural product synthesis and crop engineering. The plant cell wall is a barrier that limits the ease and throughput of exogenous biomolecule delivery to plants. Current delivery methods either suffer from host-range limitations, low transformation efficiencies, tissue damage or unavoidable DNA Integration into the host genome. Here, we demonstrate efficient diffusion-based biomolecule delivery into intact plants of several species with pristine and chemically functionalized high aspect ratio nanomaterials. Efficient DNA delivery and strong protein expression without transgene Integration is accomplished in Nicotiana benthamiana ( Nb ), Eruca sativa (arugula), Triticum aestivum (wheat) and Gossypium hirsutum (cotton) leaves and arugula protoplasts. We find that nanomaterials not only facilitate biomolecule transport into plant cells but also protect polynucleotides from nuclease degradation. Our work provides a tool for species-independent and passive delivery of genetic material, without transgene Integration, into plant cells for diverse biotechnology applications. High aspect ratio nanomaterials enable efficient delivery of DNA into mature plant cells in a species-independent and non-integrating manner for plant genetic engineering applications.

  • high aspect ratio nanomaterials enable delivery of functional genetic material without DNA Integration in mature plants
    Nature Nanotechnology, 2019
    Co-Authors: Gozde S Demirer, Huan Zhang, Juliana L Matos, Roger Chang, Linda Chio, Abhishek J Aditham, Francis J. Cunningham, Natalie Goh, Younghun Sung, Myeongje Cho
    Abstract:

    Genetic engineering of plants is at the core of sustainability efforts, natural product synthesis and crop engineering. The plant cell wall is a barrier that limits the ease and throughput of exogenous biomolecule delivery to plants. Current delivery methods either suffer from host-range limitations, low transformation efficiencies, tissue damage or unavoidable DNA Integration into the host genome. Here, we demonstrate efficient diffusion-based biomolecule delivery into intact plants of several species with pristine and chemically functionalized high aspect ratio nanomaterials. Efficient DNA delivery and strong protein expression without transgene Integration is accomplished in Nicotiana benthamiana (Nb), Eruca sativa (arugula), Triticum aestivum (wheat) and Gossypium hirsutum (cotton) leaves and arugula protoplasts. We find that nanomaterials not only facilitate biomolecule transport into plant cells but also protect polynucleotides from nuclease degradation. Our work provides a tool for species-independent and passive delivery of genetic material, without transgene Integration, into plant cells for diverse biotechnology applications.

  • high aspect ratio nanomaterials enable delivery of functional genetic material without DNA Integration in mature plants
    bioRxiv, 2018
    Co-Authors: Gozde S Demirer, Huan Zhang, Juliana L Matos, Roger Chang, Linda Chio, Abhishek J Aditham, Francis J. Cunningham, Natalie Goh, Younghun Sung, Myeongje Cho
    Abstract:

    Genetic engineering of plants is at the core of sustainability efforts, natural product synthesis, and agricultural crop engineering. The plant cell wall is a barrier that limits the ease and throughput with which exogenous biomolecules can be delivered to plants. Current delivery methods either suffer from host range limitations, low transformation efficiencies, tissue damage, or unavoidable DNA Integration into the host genome. Here, we demonstrate efficient diffusion-based biomolecule delivery into tissues and organs of intact plants of several species with a suite of pristine and chemically-functionalized high aspect ratio nanomaterials. Efficient DNA delivery and strong protein expression without transgene Integration is accomplished in Nicotiana benthamiana (Nb), Eruca sativa (arugula), Triticum aestivum (wheat) and Gossypium hirsutum (cotton) leaves and arugula protoplasts. We also demonstrate a second nanoparticle-based strategy in which small interfering RNA (siRNA) is delivered to Nb leaves and silence a gene with 95% efficiency. We find that nanomaterials not only facilitate biomolecule transport into plant cells but also protect polynucleotides from nuclease degradation. Our work provides a tool for species-independent and passive delivery of genetic material, without transgene Integration, into plant cells for diverse biotechnology applications.

  • Optimized Agrobacterium-mediated sorghum transformation protocol and molecular data of transgenic sorghum plants
    In Vitro Cellular & Developmental Biology - Plant, 2014
    Co-Authors: Brian Lenderts, Myeongje Cho, Kimberly Glassman, Maya Berezowska-kaniewska, Heather Christensen, Tracy Asmus, Shifu Zhen, Uyen Chu, Zuo-yu Zhao
    Abstract:

    Agrobacterium -mediated sorghum transformation frequency has been enhanced significantly via medium optimization using immature embryos from sorghum variety TX430 as the target tissue. The new transformation protocol includes the addition of elevated copper sulfate and 6-benzylaminopurine in the resting and selection media. Using Agrobacterium strain LBA4404, the transformation frequency reached over 10% using either of two different selection marker genes, moPAT or PMI, and any of three different vectors in large-scale transformation experiments. With Agrobacterium strain AGL1, the transformation frequencies were as high as 33%. Using quantitative PCR analyses of 1,182 T_0 transgenic plants representing 675 independent transgenic events, data was collected for T-DNA copy number, intact or truncated T-DNA Integration, and vector backbone Integration into the sorghum genome. A comparison of the transformation frequencies and molecular data characterizing T-DNA Integration patterns in the transgenic plants derived from LBA4404 versus AGL1 transformation revealed that twice as many transgenic high-quality events were generated when AGL1 was used compared to LBA4404. This is the first report providing molecular data for T-DNA Integration patterns in a large number of independent transgenic plants in sorghum.

Frederic D Bushman - One of the best experts on this subject based on the ideXlab platform.

  • ROC Curves as a Measure of the Association of DNA Integration with Genomic Features
    2013
    Co-Authors: Charles Berry, Sridhar Hannenhalli, Jeremy Leipzig, Frederic D Bushman
    Abstract:

    (A) Diagram of the ROC analysis. The graph plots the true positive rate against the false positive rate for every possible cutpoint; vertical steps result when only the true positive rate increases as the cutpoint (i.e., cutoff value for the genomic feature) moves down; horizontal steps result when only the false positive rate increases, and when both rates increase as the cutpoint moves down the graph “steps” diagonally. The example shows the effects of score.20 on SB Integration (though the method of construction is general). The area between the curve and the “no discrimination” line indicates discrimination between Integration sites and random controls by the predictor tested. The curve will lie beneath the line of “no discrimination”—leading to an area of less than 0—if Integration sites tend to have lower values of the variable under study than random controls. For details see the text and Text S1.(B) Box plots summarizing ROC results. Each box in Figure 1B indicates the first and third quartiles of the values, while the heavy line in the middle gives the median value. The “whiskers” extend to the most extreme observation within 1.5× the interquartile range of the median. Points that lie beyond the whiskers are plotted individually. For each box plot, the number of points is 17 (the number of datasets) times the number of rows in the relevant heat map for that feature (in Text S2; selected examples of heat maps are shown in Figures 2–4). Specifically, the numbers of points were 170 for gene.exon, 1173 for gene.density, 153 for DNAse, 306 for cpg, 340 for juxtapos, 1870 for transfac, 17 for score.20.all, and 340 for score.20.1.bp.

  • hiv DNA Integration
    Cold Spring Harbor Perspectives in Medicine, 2012
    Co-Authors: Robert Craigie, Frederic D Bushman
    Abstract:

    Retroviruses are distinguished from other viruses by two characteristic steps in the viral replication cycle. The first is reverse transcription, which results in the production of a double-stranded DNA copy of the viral RNA genome, and the second is Integration, which results in covalent attachment of the DNA copy to host cell DNA. The initial catalytic steps of the Integration reaction are performed by the virus-encoded integrase (IN) protein. The chemistry of the IN-mediated DNA breaking and joining steps is well worked out, and structures of IN-DNA complexes have now clarified how the overall complex assembles. Methods developed during these studies were adapted for identification of IN inhibitors, which received FDA approval for use in patients in 2007. At the chromosomal level, HIV Integration is strongly favored in active transcription units, which may promote efficient viral gene expression after Integration. HIV IN binds to the cellular factor LEDGF/p75, which promotes efficient infection and tethers IN to favored target sites. The HIV Integration machinery must also interact with many additional host factors during infection, including nuclear trafficking and pore proteins during nuclear entry, histones during initial target capture, and DNA repair proteins during completion of the DNA joining steps. Models for some of the molecular mechanisms involved have been proposed, but important details remain to be clarified.

  • selection of target sites for mobile DNA Integration in the human genome
    PLOS Computational Biology, 2006
    Co-Authors: Charles C Berry, Sridhar Hannenhalli, Jeremy Leipzig, Frederic D Bushman
    Abstract:

    DNA sequences from retroviruses, retrotransposons, DNA transposons, and parvoviruses can all become integrated into the human genome. Accumulation of such sequences accounts for at least 40% of our genome today. These integrating elements are also of interest as gene-delivery vectors for human gene therapy. Here we present a comprehensive bioinformatic analysis of Integration targeting by HIV, MLV, ASLV, SFV, L1, SB, and AAV. We used a mathematical method which allowed annotation of each base pair in the human genome for its likelihood of hosting an Integration event by each type of element, taking advantage of more than 200 types of genomic annotation. This bioinformatic resource documents a wealth of new associations between genomic features and Integration targeting. The study also revealed that the length of genomic intervals analyzed strongly affected the conclusions drawn—thus, answering the question “What genomic features affect Integration?” requires carefully specifying the length scale of interest.

  • a role for ledgf p75 in targeting hiv DNA Integration
    Nature Medicine, 2005
    Co-Authors: Angela Ciuffi, Jeremy Leipzig, Christian Hoffmann, Manuel Llano, Eric M Poeschla, Paul Shinn, Joseph R Ecker, Frederic D Bushman
    Abstract:

    HIV DNA Integration is favored in active genes, but the underlying mechanism is unclear. Cellular lens epithelium-derived growth factor (LEDGF/p75) binds both chromosomal DNA and HIV integrase, and might therefore direct Integration by a tethering interaction. We analyzed HIV Integration in cells depleted for LEDGF/p75, and found that Integration was (i) less frequent in transcription units, (ii) less frequent in genes regulated by LEDGF/p75 and (iii) more frequent in GC-rich DNA. LEDGF is thus the first example of a cellular protein controlling the location of HIV Integration in human cells.

  • genome wide analysis of retroviral DNA Integration
    Nature Reviews Microbiology, 2005
    Co-Authors: Frederic D Bushman, Mary K Lewinski, Angela Ciuffi, Stephen D Barr, Jeremy Leipzig, Sridhar Hannenhalli, Christian Hoffmann
    Abstract:

    Retroviral vectors are often used to introduce therapeutic sequences into patients' cells. In recent years, gene therapy with retroviral vectors has had impressive therapeutic successes, but has also resulted in three cases of leukaemia caused by insertional mutagenesis, which has focused attention on the molecular determinants of retroviral-Integration target-site selection. Here, we review retroviral DNA Integration, with emphasis on recent genome-wide studies of targeting and on the status of efforts to modulate target-site selection.