The Experts below are selected from a list of 309 Experts worldwide ranked by ideXlab platform
Fred Dyda - One of the best experts on this subject based on the ideXlab platform.
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structures of iscth4 transpososomes reveal the role of asymmetry in copy out paste in DNA Transposition
The EMBO Journal, 2021Co-Authors: Dalibor Kosek, Alison B Hickman, Rodolfo Ghirlando, Fred DydaAbstract:Copy-out/paste-in Transposition is a major bacterial DNA mobility pathway. It contributes significantly to the emergence of antibiotic resistance, often by upregulating expression of downstream genes upon integration. Unlike other Transposition pathways, it requires both asymmetric and symmetric strand transfer steps. Here, we report the first structural study of a copy-out/paste-in transposase and demonstrate its ability to catalyze all pathway steps in vitro. X-ray structures of ISCth4 transposase, a member of the IS256 family of insertion sequences, bound to DNA substrates corresponding to three sequential steps in the reaction reveal an unusual asymmetric dimeric transpososome. During Transposition, an array of N-terminal domains binds a single transposon end while the catalytic domain moves to accommodate the varying substrates. These conformational changes control the path of DNA flanking the transposon end and the generation of DNA-binding sites. Our results explain the asymmetric outcome of the initial strand transfer and show how DNA binding is modulated by the asymmetric transposase to allow the capture of a second transposon end and to integrate a circular intermediate.
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Structures of ISCth4 transpososomes reveal the role of asymmetry in copy-out/paste-in DNA Transposition.
The EMBO journal, 2020Co-Authors: Dalibor Kosek, Alison B Hickman, Rodolfo Ghirlando, Fred DydaAbstract:Copy-out/paste-in Transposition is a major bacterial DNA mobility pathway. It contributes significantly to the emergence of antibiotic resistance, often by upregulating expression of downstream genes upon integration. Unlike other Transposition pathways, it requires both asymmetric and symmetric strand transfer steps. Here, we report the first structural study of a copy-out/paste-in transposase and demonstrate its ability to catalyze all pathway steps in vitro. X-ray structures of ISCth4 transposase, a member of the IS256 family of insertion sequences, bound to DNA substrates corresponding to three sequential steps in the reaction reveal an unusual asymmetric dimeric transpososome. During Transposition, an array of N-terminal domains binds a single transposon end while the catalytic domain moves to accommodate the varying substrates. These conformational changes control the path of DNA flanking the transposon end and the generation of DNA-binding sites. Our results explain the asymmetric outcome of the initial strand transfer and show how DNA binding is modulated by the asymmetric transposase to allow the capture of a second transposon end and to integrate a circular intermediate.
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casposase structure and the mechanistic link between DNA Transposition and spacer acquisition by crispr cas
eLife, 2020Co-Authors: Alison B Hickman, Shweta Kailasan, Pavol Genzor, Astrid D Haase, Fred DydaAbstract:: Key to CRISPR-Cas adaptive immunity is maintaining an ongoing record of invading nucleic acids, a process carried out by the Cas1-Cas2 complex that integrates short segments of foreign genetic material (spacers) into the CRISPR locus. It is hypothesized that Cas1 evolved from casposases, a novel class of transposases. We show here that the Methanosarcina mazei casposase can integrate varied forms of the casposon end in vitro, and recapitulates several properties of CRISPR-Cas integrases including site-specificity. The X-ray structure of the casposase bound to DNA representing the product of integration reveals a tetramer with target DNA bound snugly between two dimers in which single-stranded casposon end binding resembles that of spacer 3'-overhangs. The differences between transposase and CRISPR-Cas integrase are largely architectural, and it appears that evolutionary change involved changes in protein-protein interactions to favor Cas2 binding over tetramerization; this in turn led to preferred integration of single spacers over two transposon ends.
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DNA Transposition at Work
Chemical reviews, 2016Co-Authors: Alison B Hickman, Fred DydaAbstract:DNA transposons are defined segments of DNA that are able to move from one genomic location to another. Movement is facilitated by one or more proteins, called the transposase, typically encoded by the mobile element itself. Here, we first provide an overview of the classification of such mobile elements in a variety of organisms. From a mechanistic perspective, we have focused on one particular group of DNA transposons that encode a transposase with a DD(E/D) catalytic domain that is topologically similar to RNase H. For these, a number of three-dimensional structures of transpososomes (transposase–nucleic acid complexes) are available, and we use these to describe the basics of their mechanisms. The DD(E/D) group, in addition to being the largest and most common among all DNA transposases, is the one whose members have been used for a wide variety of genomic applications. Therefore, a second focus of the article is to provide a nonexhaustive overview of transposon applications. Although several non-tran...
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mechanism of spacer integration links the crispr cas system to Transposition as a form of mobile DNA
Mobile Dna, 2015Co-Authors: Fred Dyda, Alison B HickmanAbstract:It has recently become clear that many bacterial and archaeal species possess adaptive immune systems. These are typified by multiple copies of DNA sequences known as clustered regularly interspaced short palindromic repeats (CRISPRs). These CRISPR repeats are the sites at which short spacers containing sequences of previously encountered foreign DNA are integrated, and the spacers serve as the molecular memory of previous invaders. In vivo work has demonstrated that two CRISPR-associated proteins - Cas1 and Cas2 - are required for spacer integration, but the mechanism by which this is accomplished remained unclear. Here we review a recent paper describing the in vitro reconstitution of CRISPR spacer integration using purified Cas1 and Cas2 and place the results in context of similar DNA Transposition reactions and the crystal structure of the Cas1/Cas2 complex.
Harri Savilahti - One of the best experts on this subject based on the ideXlab platform.
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Mu transpososome activity-profiling yields hyperactive MuA variants for highly efficient genetic and genome engineering.
Nucleic acids research, 2017Co-Authors: Tiina S Rasila, Saija Haapa-paananen, Saija Kiljunen, Lars Paulin, Elsi Pulkkinen, Mauno Vihinen, Maria Pajunen, Anu Salminen, Phoebe A. Rice, Harri SavilahtiAbstract:The phage Mu DNA Transposition system provides a versatile species non-specific tool for molecular biology, genetic engineering and genome modification applications. Mu Transposition is catalyzed by MuA transposase, with DNA cleavage and integration reactions ultimately attaching the transposon DNA to target DNA. To improve the activity of the Mu DNA Transposition machinery, we mutagenized MuA protein and screened for hyperactivity-causing substitutions using an in vivo assay. The individual activity-enhancing substitutions were mapped onto the MuA-DNA complex structure, containing a tetramer of MuA transposase, two Mu end segments and a target DNA. This analysis, combined with the varying effect of the mutations in different assays, implied that the mutations exert their effects in several ways, including optimizing protein-protein and protein-DNA contacts. Based on these insights, we engineered highly hyperactive versions of MuA, by combining several synergistically acting substitutions located in different subdomains of the protein. Purified hyperactive MuA variants are now ready for use as second-generation tools in a variety of Mu-based DNA Transposition applications. These variants will also widen the scope of Mu-based gene transfer technologies toward medical applications such as human gene therapy. Moreover, the work provides a platform for further design of custom transposases.
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Applications of the Bacteriophage Mu In Vitro Transposition Reaction and Genome Manipulation via Electroporation of DNA Transposition Complexes.
Methods in molecular biology (Clifton N.J.), 2017Co-Authors: Saija Haapa-paananen, Harri SavilahtiAbstract:The capacity of transposable elements to insert into the genomes has been harnessed during the past decades to various in vitro and in vivo applications. This chapter describes in detail the general protocols and principles applicable for the Mu in vitro Transposition reaction as well as the assembly of DNA Transposition complexes that can be electroporated into bacterial cells to accomplish efficient gene delivery. These techniques with their modifications potentiate various gene and genome modification applications, which are discussed briefly here, and the reader is referred to the original publications for further details.
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An assay to monitor the activity of DNA Transposition complexes yields a general quality control measure for Transpositional recombination reactions.
Mobile genetic elements, 2014Co-Authors: Elsi Pulkkinen, Saija Haapa-paananen, Harri SavilahtiAbstract:Transposon-based technologies have many applications in molecular biology and can be used for gene delivery into prokaryotic and eukaryotic cells. Common Transpositional activity measurement assays suitable for many types of transposons would be beneficial, as diverse transposon systems could be compared for their performance attributes. Therefore, we developed a general-purpose assay to enable and standardize the activity measurement for DNA Transposition complexes (transpososomes), using phage Mu Transposition as a test platform. This assay quantifies Transpositional recombination efficiency and is based on an in vitro Transposition reaction with a target plasmid carrying a lethal ccdB gene. If Transposition targets ccdB, this gene becomes inactivated, enabling plasmid-receiving Escherichia coli cells to survive and to be scored as colonies on selection plates. The assay was validated with 3 mini-Mu transposons varying in size and differing in their marker gene constitution. Tests with different amounts of transposon DNA provided a linear response and yielded a 10-fold operational range for the assay. The colony formation capacity was linearly correlated with the competence status of the E.coli cells, enabling normalization of experimental data obtained with different batches of recipient cells. The developed assay can now be used to directly compare transpososome activities with all types of mini-Mu transposons, regardless of their aimed use. Furthermore, the assay should be directly applicable to other Transposition-based systems with a functional in vitro reaction, and it provides a dependable quality control measure that previously has been lacking but is highly important for the evaluation of current and emerging transposon-based applications.
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Universal platform for quantitative analysis of DNA Transposition.
Mobile DNA, 2010Co-Authors: Maria Pajunen, Tiina S Rasila, Lotta Happonen, Arja Lamberg, Saija Haapa-paananen, Saija Kiljunen, Harri SavilahtiAbstract:Background Completed genome projects have revealed an astonishing diversity of transposable genetic elements, implying the existence of novel element families yet to be discovered from diverse life forms. Concurrently, several better understood transposon systems have been exploited as efficient tools in molecular biology and genomics applications. Characterization of new mobile elements and improvement of the existing Transposition technology platforms warrant easy-to-use assays for the quantitative analysis of DNA Transposition.
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Universal platform for quantitative analysis of DNA Transposition
Mobile DNA, 2010Co-Authors: Maria Pajunen, Tiina S Rasila, Lotta Happonen, Arja Lamberg, Saija Haapa-paananen, Saija Kiljunen, Harri SavilahtiAbstract:Background Completed genome projects have revealed an astonishing diversity of transposable genetic elements, implying the existence of novel element families yet to be discovered from diverse life forms. Concurrently, several better understood transposon systems have been exploited as efficient tools in molecular biology and genomics applications. Characterization of new mobile elements and improvement of the existing Transposition technology platforms warrant easy-to-use assays for the quantitative analysis of DNA Transposition. Results Here we developed a universal in vivo platform for the analysis of Transposition frequency with class II mobile elements, i.e., DNA transposons. For each particular transposon system, cloning of the transposon ends and the cognate transposase gene, in three consecutive steps, generates a multifunctional plasmid, which drives inducible expression of the transposase gene and includes a mobilisable lacZ -containing reporter transposon. The assay scores Transposition events as blue microcolonies, papillae, growing within otherwise whitish Escherichia coli colonies on indicator plates. We developed the assay using phage Mu Transposition as a test model and validated the platform using various MuA transposase mutants. For further validation and to illustrate universality, we introduced IS 903 Transposition system components into the assay. The developed assay is adjustable to a desired level of initial Transposition via the control of a plasmid-borne E. coli arabinose promoter. In practice, the Transposition frequency is modulated by varying the concentration of arabinose or glucose in the growth medium. We show that variable levels of Transpositional activity can be analysed, thus enabling straightforward screens for hyper- or hypoactive transposase mutants, regardless of the original wild-type activity level. Conclusions The established universal papillation assay platform should be widely applicable to a variety of mobile elements. It can be used for mechanistic studies to dissect Transposition and provides a means to screen or scrutinise transposase mutants and genes encoding host factors. In succession, improved versions of Transposition systems should yield better tools for molecular biology and offer versatile genome modification vehicles for many types of studies, including gene therapy and stem cell research.
Alison B Hickman - One of the best experts on this subject based on the ideXlab platform.
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structures of iscth4 transpososomes reveal the role of asymmetry in copy out paste in DNA Transposition
The EMBO Journal, 2021Co-Authors: Dalibor Kosek, Alison B Hickman, Rodolfo Ghirlando, Fred DydaAbstract:Copy-out/paste-in Transposition is a major bacterial DNA mobility pathway. It contributes significantly to the emergence of antibiotic resistance, often by upregulating expression of downstream genes upon integration. Unlike other Transposition pathways, it requires both asymmetric and symmetric strand transfer steps. Here, we report the first structural study of a copy-out/paste-in transposase and demonstrate its ability to catalyze all pathway steps in vitro. X-ray structures of ISCth4 transposase, a member of the IS256 family of insertion sequences, bound to DNA substrates corresponding to three sequential steps in the reaction reveal an unusual asymmetric dimeric transpososome. During Transposition, an array of N-terminal domains binds a single transposon end while the catalytic domain moves to accommodate the varying substrates. These conformational changes control the path of DNA flanking the transposon end and the generation of DNA-binding sites. Our results explain the asymmetric outcome of the initial strand transfer and show how DNA binding is modulated by the asymmetric transposase to allow the capture of a second transposon end and to integrate a circular intermediate.
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Structures of ISCth4 transpososomes reveal the role of asymmetry in copy-out/paste-in DNA Transposition.
The EMBO journal, 2020Co-Authors: Dalibor Kosek, Alison B Hickman, Rodolfo Ghirlando, Fred DydaAbstract:Copy-out/paste-in Transposition is a major bacterial DNA mobility pathway. It contributes significantly to the emergence of antibiotic resistance, often by upregulating expression of downstream genes upon integration. Unlike other Transposition pathways, it requires both asymmetric and symmetric strand transfer steps. Here, we report the first structural study of a copy-out/paste-in transposase and demonstrate its ability to catalyze all pathway steps in vitro. X-ray structures of ISCth4 transposase, a member of the IS256 family of insertion sequences, bound to DNA substrates corresponding to three sequential steps in the reaction reveal an unusual asymmetric dimeric transpososome. During Transposition, an array of N-terminal domains binds a single transposon end while the catalytic domain moves to accommodate the varying substrates. These conformational changes control the path of DNA flanking the transposon end and the generation of DNA-binding sites. Our results explain the asymmetric outcome of the initial strand transfer and show how DNA binding is modulated by the asymmetric transposase to allow the capture of a second transposon end and to integrate a circular intermediate.
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casposase structure and the mechanistic link between DNA Transposition and spacer acquisition by crispr cas
eLife, 2020Co-Authors: Alison B Hickman, Shweta Kailasan, Pavol Genzor, Astrid D Haase, Fred DydaAbstract:: Key to CRISPR-Cas adaptive immunity is maintaining an ongoing record of invading nucleic acids, a process carried out by the Cas1-Cas2 complex that integrates short segments of foreign genetic material (spacers) into the CRISPR locus. It is hypothesized that Cas1 evolved from casposases, a novel class of transposases. We show here that the Methanosarcina mazei casposase can integrate varied forms of the casposon end in vitro, and recapitulates several properties of CRISPR-Cas integrases including site-specificity. The X-ray structure of the casposase bound to DNA representing the product of integration reveals a tetramer with target DNA bound snugly between two dimers in which single-stranded casposon end binding resembles that of spacer 3'-overhangs. The differences between transposase and CRISPR-Cas integrase are largely architectural, and it appears that evolutionary change involved changes in protein-protein interactions to favor Cas2 binding over tetramerization; this in turn led to preferred integration of single spacers over two transposon ends.
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DNA Transposition at Work
Chemical reviews, 2016Co-Authors: Alison B Hickman, Fred DydaAbstract:DNA transposons are defined segments of DNA that are able to move from one genomic location to another. Movement is facilitated by one or more proteins, called the transposase, typically encoded by the mobile element itself. Here, we first provide an overview of the classification of such mobile elements in a variety of organisms. From a mechanistic perspective, we have focused on one particular group of DNA transposons that encode a transposase with a DD(E/D) catalytic domain that is topologically similar to RNase H. For these, a number of three-dimensional structures of transpososomes (transposase–nucleic acid complexes) are available, and we use these to describe the basics of their mechanisms. The DD(E/D) group, in addition to being the largest and most common among all DNA transposases, is the one whose members have been used for a wide variety of genomic applications. Therefore, a second focus of the article is to provide a nonexhaustive overview of transposon applications. Although several non-tran...
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mechanism of spacer integration links the crispr cas system to Transposition as a form of mobile DNA
Mobile Dna, 2015Co-Authors: Fred Dyda, Alison B HickmanAbstract:It has recently become clear that many bacterial and archaeal species possess adaptive immune systems. These are typified by multiple copies of DNA sequences known as clustered regularly interspaced short palindromic repeats (CRISPRs). These CRISPR repeats are the sites at which short spacers containing sequences of previously encountered foreign DNA are integrated, and the spacers serve as the molecular memory of previous invaders. In vivo work has demonstrated that two CRISPR-associated proteins - Cas1 and Cas2 - are required for spacer integration, but the mechanism by which this is accomplished remained unclear. Here we review a recent paper describing the in vitro reconstitution of CRISPR spacer integration using purified Cas1 and Cas2 and place the results in context of similar DNA Transposition reactions and the crystal structure of the Cas1/Cas2 complex.
William S Reznikoff - One of the best experts on this subject based on the ideXlab platform.
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Transposon Tn5.
Annual review of genetics, 2008Co-Authors: William S ReznikoffAbstract:Tn5 was one of the first transposons to be identified ( 10 ). As a result of Tn5's early discovery and its simple macromolecular requirements for Transposition, the Tn5 system has been a very productive tool for studying the molecular mechanism of DNA Transposition. These studies are of broad value because they offer insights into DNA Transposition in general, because DNA Transposition is a useful model with which to understand other types of protein-DNA interactions such as retroviral DNA integration and the DNA cleavage events involved in immunoglobulin gene formation, and because Tn5-derived tools are useful adjuncts in genetic experimentation.
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Tn5 as a model for understanding DNA Transposition
Molecular microbiology, 2003Co-Authors: William S ReznikoffAbstract:Tn5 is an excellent model system for understanding the molecular basis of DNA-mediated Transposition. Mechanistic information has come from genetic and biochemical investigations of the transposase and its interactions with the recognition DNA sequences at the ends of the transposon. More recently, molecular structure analyses of catalytically active transposase; transposon DNA complexes have provided us with unprecedented insights into this Transposition system. Transposase initiates Transposition by forming a dimeric transposase, transposon DNA complex. In the context of this complex, the transposase then catalyses four phosphoryl transfer reactions (DNA nicking, DNA hairpin formation, hairpin resolution and strand transfer into target DNA) resulting in the integration of the transposon into its new DNA site. The studies that elucidated these steps also provided important insights into the integration of retroviral genomes into host DNA and the immune system V(D)J joining process. This review will describe the structures and steps involved in Tn5 Transposition and point out a biologically important although surprising characteristic of the wild-type Tn5 transposase. Transposase is a very inactive protein. An inactive transposase protein ensures the survival of the host and thus the survival of Tn5.
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eLS - DNA Transposition: Classes and Mechanisms
Encyclopedia of Life Sciences, 2001Co-Authors: Lisa Am Braam, William S ReznikoffAbstract:Transposons are mobile genetic elements that contribute to the dynamic nature of chromosomes. Mechanistically similar, transposable elements make up a diverse classification including bacterial insertion sequences and composite transposons and eukaryotic excision/insertion transposons and retroelements. Keywords: transposon; retrotransposon; retrovirus; transfer; integration
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three dimensional structure of the tn5 synaptic complex Transposition intermediate
Science, 2000Co-Authors: Douglas R Davies, Igor Y Goryshin, William S Reznikoff, Ivan RaymentAbstract:Genomic evolution has been profoundly influenced by DNA Transposition, a process whereby defined DNA segments move freely about the genome. Transposition is mediated by transposases, and similar events are catalyzed by retroviral integrases such as human immunodeficiency virus–1 (HIV-1) integrase. Understanding how these proteins interact with DNA is central to understanding the molecular basis of Transposition. We report the three-dimensional structure of prokaryotic Tn 5 transposase complexed with Tn 5 transposon end DNA determined to 2.3 angstrom resolution. The molecular assembly is dimeric, where each double-stranded DNA molecule is bound by both protein subunits, orienting the transposon ends into the active sites. This structure provides a molecular framework for understanding many aspects of Transposition, including the binding of transposon end DNA by one subunit and cleavage by a second, cleavage of two strands of DNA by a single active site via a hairpin intermediate, and strand transfer into target DNA.
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insertional transposon mutagenesis by electroporation of released tn5 Transposition complexes
Nature Biotechnology, 2000Co-Authors: Igor Y Goryshin, Jerry Jendrisak, Les M Hoffman, Ronald Meis, William S ReznikoffAbstract:DNA Transposition is an important biological phenomenon that mediates genome rearrangements, inheritance of antibiotic resistance determinants, and integration of retroviral DNA. Transposition has also become a powerful tool in genetic analysis, with applications in creating insertional knockout mutations, generating gene–operon fusions to reporter functions, providing physical or genetic landmarks for the cloning of adjacent DNAs, and locating primer binding sites for DNA sequence analysis. DNA Transposition studies to date usually have involved strictly in vivo approaches, in which the transposon of choice and the gene encoding the transposase responsible for catalyzing the Transposition have to be introduced into the cell to be studied (microbial systems and applications are reviewed in ref. 1). However, all in vivo systems have a number of technical limitations. For instance, the transposase must be expressed in the target host, the transposon must be introduced into the host on a suicide vector, and the transposase usually is expressed in subsequent generations, resulting in potential genetic instability. A number of in vitro Transposition systems (for Tn5, Tn7, Mu, Himar1 , and Ty1) have been described, which bypass many limitations of in vivo systems2,3,4,5,6,7. For this purpose, we have developed a technique for Transposition that involves the formation in vitro of released Tn5 Transposition complexes (TransposomesTM) followed by introduction of the complexes into the target cell of choice by electroporation. In this report, we show that this simple, robust technology can generate high-efficiency Transposition in all tested bacterial species (Escherichia coli, Salmonella typhimurium, and Proteus vulgaris) We also isolated Transposition events in the yeast Saccharomyces cerevisiae.
George Chaconas - One of the best experts on this subject based on the ideXlab platform.
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3D reconstruction of the Mu transposase and the Type 1 transpososome: a structural framework for Mu DNA Transposition
Genes & development, 2005Co-Authors: Joy F. Yuan, George Chaconas, Daniel R. Beniac, F. Peter OttensmeyerAbstract:Mu DNA Transposition proceeds through a series of higher-order nucleoprotein complexes called transpososomes. The structural core of the transpososome is a tetramer of the transposase, Mu A, bound to the two transposon ends. High-resolution structural analysis of the intact transposase and the transpososome has not been successful to date. Here we report the structure of Mu A at 16-A and the Type 1 transpososome at 34-A resolution, by 3D reconstruction of images obtained by scanning transmission electron microscopy (STEM) at cryo-temperatures. Electron spectroscopic imaging (ESI) of the DNA-phosphorus was performed in conjunction with the structural investigation to derive the path of the DNA through the transpososome and to define the DNA-binding surface in the transposase. Our model of the transpososome fits well with the accumulated biochemical literature for this intricate Transposition system, and lays a structural foundation for biochemical function, including catalysis in trans and the complex circuit of macromolecular interactions underlying Mu DNA Transposition.
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Studies on a "Jumping Gene Machine": Higher-Order Nucleoprotein Complexes in
2000Co-Authors: George ChaconasAbstract:Studies in my lab have focused on DNA Transposition in the bacterial virus, Mu. In vitro studies have shown that Mu DNA Transposition is a three-step process involving DNA breakage, strand transfer and DNA replication. In the first step a nick is introduced at each end of the transposon. The liberated 3'-OH groups subsequently attack a target DNA molecule resulting in strand transfer. The transposon DNA, now covalently linked to the target, is finally replicated to generate the Transposition end-product, referred to as a cointegrate. The DNA cleavage and strand transfer reactions are mediated by a “jumping gene machine” or transpososomes, which we discovered in 1987. They are assembled by bringing together three different DNA regions via a process involving multiple proteinDNA and protein-protein interactions. The action of four different proteins is required in addition to protein-induced DNA bending or wrapping to overcome the intrinsic stiffness of DNA, which would ordinarily prohibit the assembly of such a structure. Transpososome assembly is a gradual process involving multiple steps with an inherent flexibility whereby alternate pathways can be used in the assembly process, biasing the reaction towards completion under different conditions.
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Studies on a "jumping gene machine": higher-order nucleoprotein complexes in Mu DNA Transposition.
Biochemistry and cell biology = Biochimie et biologie cellulaire, 1999Co-Authors: George ChaconasAbstract:Studies in my lab have focused on DNA Transposition in the bacterial virus, Mu. In vitro studies have shown that Mu DNA Transposition is a three-step process involving DNA breakage, strand transfer and DNA replication. In the first step, a nick is introduced at each end of the transposon. The liberated 3'-OH groups subsequently attack a target DNA molecule resulting in strand transfer. The transposon DNA, now covalently linked to the target, is finally replicated to generate the Transposition end-product, referred to as a cointegrate. The DNA cleavage and strand transfer reactions are mediated by a "jumping gene machine" or transpososomes, which we discovered in 1987. They are assembled by bringing together three different DNA regions via a process involving multiple protein-DNA and protein-protein interactions. The action of four different proteins is required in addition to protein-induced DNA bending or wrapping to overcome the intrinsic stiffness of DNA, which would ordinarily prohibit the assembly of such a structure. Transpososome assembly is a gradual process involving multiple steps with an inherent flexibility whereby alternate pathways can be used in the assembly process, biasing the reaction towards completion under different conditions.
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A new set of Mu DNA Transposition intermediates: alternate pathways of target capture preceding strand transfer.
The EMBO journal, 1997Co-Authors: Darius Z. Naigamwalla, George ChaconasAbstract:Mu DNA Transposition occurs within the context of higher order nucleoprotein structures or transpososomes. We describe a new set of transpososomes in which Mu B-bound target DNA interacts non-covalently with previously characterized intermediates prior to the actual strand transfer. This interaction can occur at several points along the reaction pathway: with the LER, the Type 0 or the Type 1 complexes. The formation of these target capture complexes, which rapidly undergo the strand transfer chemistry, is the rate-limiting step in the overall reaction. These complexes provide alternate pathways to strand transfer, thereby maximizing Transposition potential. This versatility is in contrast to other characterized transposons, which normally capture target DNA only at a single point in their respective reaction pathways.
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DNA Transposition: Assembly of a jumping gene machine
Current biology : CB, 1996Co-Authors: George Chaconas, Brigitte Lavoie, Mark A WatsonAbstract:Abstract Transposition of the mobile DNA element Mu is stringently controlled by the assembly of an elaborate ‘jumping gene machine', which is inactive until all the pieces are in place.