Browsing by Subject "Transposons"
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Item Identification of two topologically distinct Mu transpososomes: contribution of cis and trans elements to DNA topology(2006) Yin, Zhiqi; Harshey, Rasika M.Transposition of bacteriophage Mu takes place within nucleoprotein complexes called transpososomes. Assembly of a transpososome requires transposase binding to multiple sites within the L and R ends of Mu and an internal transpositional enhancer E, present on supercoiled DNA. L, R and E interact such that five negative DNA supercoils are trapped within the transpososome. We have carried out topological studies aimed at understanding the contribution of all of these elements to transpososome assembly. We have found that the transposase has an inherent capacity to interwrap distant DNA sites. Under enhancer-independent reaction conditions, two topologically distinct synapses were identified. These studies have revealed that the enhancer as well as the ends, contribute to the topological selectivity of the Mu synapse. We have identified specific end – enhancer interactions critical for assembly. Based on our topological studies as well as other data, we propose a new model for the structure of the 5-noded 3-site Mu complex.Item Importance of the conserved TG/CA dinucleotide termini in phage Mu transposition: similarities to transposable elements in the human genome(2002) Lee, Insuk; Harshey, Rasika M.The dinucleotide TG/CA found at the termini of transposable phage Mu also occurs at the termini of a large class of transposable elements (TEs). In order to understand its importance, the activity of all 16 dinucleotide permutations of the termini was examined using a sensitive plasmid-based in vivo transposition assay. The reactivity of these substrates varied over several orders of magnitude in vivo, with substitutions at the T/A position being more severely impaired than those at the G/C position. The same general heirarchy of reactivity was observed in vitro using mutant oligonucleotide substrates. These experiments revealed that TG/CA was important not for the chemistry of strand transfer but for the stage of assembly of a stable transpososome. Given that DNA at the Mu-host junctions is melted/distorted concomitantly with transpososome assembly, we hypothesized that the terminal TG/CA dinucleotide has been selected primarily for its conformational flexibility. To test this hypothesis, we examined the activity of substrates carrying a hundred different pairs of mismatched termini. Consistent with the flexibility hypothesis, we found that mismatched substrates are extremely efficient at assembly. A wild-type T residue on the bottom strand is essential for stable assembly, but the identity of the dinucleotide on the top strand is irrelevant for transposition chemistry. In addition, we have found a new rule for suppression of terminal defects by MuB protein, as well as a role for metal ions in DNA opening at the termini. To extend the flexibility hypothesis to other TEs that perform DNA cleavage and strand transfer at precise DNA positions, we performed a statistical analysis of sequences found at the termini of precise TEs in the human genome. The results showed that LTR retrotransposons and DNA transposons encode the most flexible dinucleotide (TG/CA) and trinucleotide (CAG/CTG) most frequently at their termini, respectively. Combining results from this statistical analysis with previous findings in phage Mu transposition, we propose that a flexible terminal 2-3 base pair step is a core component of the machinery of precise TEs, and that molecular interactions at the +1 position influence a rate-limiting step.Item Studies in bacterial genome engineering and its applications(2014-05) Enyeart, Peter James; Ellington, Andrew D.Many different approaches exist for engineering bacterial genomes. The most common current methods include transposons for random mutagenesis, recombineering for specific modifications in Escherichia coli, and targetrons for targeted knock-outs. Site-specific recombinases, which can catalyze a variety of large modifications at high efficiency, have been relatively underutilized in bacteria. Employing these technologies in combination could significantly expand and empower the toolkit available for modifying bacteria. Targetrons can be adapted to carry functional genetic elements to defined genomic loci. For instance, we re-engineered targetrons to deliver lox sites, the recognition target of the site-specific recombinase, Cre. We used this system on the E. coli genome to delete over 100 kilobases, invert over 1 megabase, insert a 12-kilobase polyketide-synthase operon, and translocate a 100 kilobase section to another site over 1 megabase away. We further used it to delete a 15-kilobase pathogenicity island from Staphylococcus aureus, catalyze an inversion of over 1 megabase in Bacillus subtilis, and simultaneously deliver nine lox sites to the genome of Shewanella oneidensis. This represents a powerful, versatile, and broad-host-range solution for bacterial genome engineering. We also placed lox sites on mariner transposons, which we leveraged to create libraries of millions of strains harboring rearranged genomes. The resulting data represents the most thorough search of the space of potential genomic rearrangements to date. While simple insertions were often most adaptive, the most successful modification found was an inversion that significantly improved fitness in minimal media. This approach could be pushed further to examine swapping or cutting and pasting regions of the genome, as well. As potential applications, we present work towards implementing and optimizing extracellular electron transfer in E. coli, as well as mathematical models of bacteria engineered to adhere to the principles of the economic concept of comparative advantage, which indicate that the approach is feasible, and furthermore indicate that economic cooperation is favored under more adverse conditions. Extracellular electron transfer has applications in bioenergy and biomechanical interfaces, while synthetic microbial economics has applications in designing consortia-based industrial bioprocesses. The genomic engineering methods presented above could be used to implement and optimize these systems.