Mechanism of Insertion Sequence Translocation

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DESCRIPTION (Provided by applicant): The overall goal of this project is to understand, in as much detail as possible, the mechanisms of two different kinds of specialized recombination associated with transposable DNA elements: site-specific recombination and transpositional recombination. The model system for site-specific recombination - a breakage-exchange-reunion reaction between two specific sites - is the resolution of cointegrates (the product of transpositional recombination by the gamma delta transposon) by the gamma delta resolvase, prototype of the serine recombinases. Transpositional recombination will be studied using a transposon of the D,D(35)E superfamily, Tn552. Site-specific recombination mediated by the gamma delta resolvase. A primary goal is to elucidate the architecture of the synaptic complex (containing two 120 bp ressites and 6 dimers of resolvase) within which recombination occurs. We have formulated a new structural model for this complex based on crystallography of the resolvase dimer and a detailed knowledge of interdimer interactions. A variety of approaches, particularly fluorescence resonance energy transfer (FRET), will be used both to test predictions of this (and an opposing) model, and also to probe for large rearrangements of the resolvase subunits or domains that accompany the activation of catalytic functions and the process of strand exchange. An investigation of another related serine recombinase, the transposase of the Helicobactor pylori element, IS60 7, will also be initiated. Transpositional recombination by Tn552. We have developed an efficient in vitro strand transfer reaction for Tn552, using the TnpA transposase and a transposon substrate with pre-cleaved ends. We intend to focus on the role of the accessory transposition protein, TnpB in activation of TnpA (allowing it to cleave uncleaved transposon ends) and in transposition immunity.