MECHANISM OF INSERTION SEQUENCE TRANSLOCATION

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The overall goal of this project is to understand, in as much molecular detail as possible, the mechanisms of two different types of specialized recombination: transpositional recombination and site-specific recombination. Transpositional recombination - the integration of a transposable DNA element into a target site - will be investigated using two contrasting transposons as model systems. These are IS903, which appears to transpose predominantly by a donor-destructive non-replicative excision-insertion pathway, and gamma delta, which uses a fully replicative pathway in which the donor survives intact. The model system for site- specific recombination - the recombination between two specific sites by a reciprocal conservative breakage-reunion reaction - is the resolution of gamma delta cointegrates (the product of gamma delta transpositional recombination) mediated by the gamma delta-encoded resolvase. I.Transpositional Recombination For IS903 and gamma delta we shall attempt to set up in vitro systems for transposition, building on our increased knowledge regarding the production and behavior of the transposase proteins. Sensitive assays, able to detect the strand transfer intermediates as well as the final products of transposition, will be used. Attempts will be made, using genetic and biochemical approaches, to identify the regions of each transposase responsible for catalytic functions and for recognition of the unusually long DNA binding sites at the transposon termini. Two transposon-specific characteristics will also be investigated: the strong cis-acting preference of the IS903 transposase and the transpositional immunity exhibited by gammadelta. II.Site-specific Recombination Mediated by the gamma delta Resolvase The primary aim is to understand the molecular nature of the two nucleoprotein complexes formed by the interaction of resolvase with res: the resolvosome, a single res bound by 3 (presumably) resolvase dimers, and the synaptosome, the complex with two paired res sites within which strand exchange occurs. The crystal structure of the catalytic domain of resolvase provides a structural basis for the objectives which are: (a to identify the dimeric arrangement of resolvase monomers responsible for the strand cleavage at each crossover point; (b) to define the various pairwise interactions between resolvase protomers that result in assembly of the resolvosome and synaptosome, determining which resolvase-bound subsites interact with one another and identifying the surfaces of interaction used; (c) to define the path of the res DNA relative to the packing of the catalytic domains. An additional aim is to understand more fully the process of DNA recognition and binding b the C-terminal domain of resolvase.