The long-term objective of this proposal is to understand the mechanisms of genetic transposition in transposable elements like IS2. Transposable elements - independent mobile genetic units, are known to play specific roles in both congenital and infectious human diseases, and they have played an important role in the evolution of vertebrate immune systems, genome evolution and thus biological evolution. IS2 belongs to the IS3 family of bacterial insertion sequences. It has a novel 2-step transposition pathway characteristic of this family that is catalyzed by the single functional recombinase of the element -its transposase. The pathway is highlighted by the formation of a circularized excised version of the element - the minicircle. The first step of the pathway (minicircle formation) is achieved by an asymmetric cleavage and joining reaction in which the right end (the exclusive donor) is abutted to the passive left end (the exclusive target) to form the mini-circle junction. In the second step (minicircle insertion) both ends become donors and are transferred to an intermolecular target, resulting in the insertion of the element at a new site. Recent in vivo genetic analysis has led to the hypothesis that each of the two proposed steps occurs in intrinsically different protein/DNA complexes - synaptic complexes or transpososomes. This novel two-transpososome model will be tested through three specific aims: (i) to obtain soluble and highly active preparations of the transposase from hyperactive mutants (ii) to develop in vitro gel retardation, and DNA footprinting analyses to examine the types of protein/DNA interactions which occur between the transposase and the free ends of the element in the minicircle formation complex and (iii) to determine whether different protein/DNA complexes are formed in a minicircle insertion complex as compared to a minicircle formation complex. Gel retardation techniques will be coupled with DNase I, hydroxyl radical, methyl interference or copper phenanthroline and permanganate DNA footprinting, to probe the specific and non-specific protein/DNA contacts in the complex(es).
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