The long term objective of this study is to understand at a mechanistic level how transposition occurs in the insertion sequence IS2. IS2 is a 1.3kb transposable element which is a member of the IS3 family of insertion sequences. Presently, nothing is known of the mechanistic details of how transposition is achieved in this group of transposable elements. This family has several features in common with other TEs at the nucleotide sequence level; in addition IS2 and other IS3-like TEs produce a transposes which catalyzes transposition and which is thought to bear striking structural and functional similarities to the integrase protein (IN) of the retroviruses. An understanding of the mechanisms by which the transposes catalyzes transposition could, therefore, contribute to an overall mechanistic and functional understanding of integrases as well.
The specific aims of this study are; (i) to develop an in vivo transpositional assay through which inter and intramolecular transposition events can be studied. This will involve the preparation of plasmid constructs with altered versions of IS2 which transpose at high frequencies and carry antibiotic resistance markers; with these constructs the genetic criteria for transposition can be readily studied using a mutational approach. (ii) to identify the DNA sequences to which transposase binds, both within the element and at preferential insertion sites (e.g., the hemB gene of E. coli). This will be achieved through gel retardation and DNA footprinting studies. (iii) to dissect the mechanistic details of transposase-mediated recombination events; these include the target and nature of its cleavage or processing reactions, the nature of strand transfer reactions which generate athe recombination product(s), and the formation of higher order DNA-protein complexes, within which the cleavage and joining reactions are thought to occur. This will be done using an in vitro transposition assay, whose reaction products will be analyzed with labelled substrates on polyacrylamide gels and by Southern hybridization techniques using agarose gels.
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