The long-term goal of this project is to understand mechanistically how transposition occurs in insertion sequences like IS2. IS2 belongs to the wide spread family of bacterial elements, in which a novel 2-step pathway of transposition has recently been described. Until now two classical paradigm described transpositional mechanisms in prokaryotic and eukaryotic elements. The identification of this new pathway in two members of the IS3 family, IS2 and IS911, has led to the general recognition of a 3rd mechanism of transposition. This lab has determined some of the mechanistic details of the 2-step transpositional pathway in IS2. In the first step, (i) transposition is initiated by a novel asymmetric single strand nick at the right end of the element only. (ii) a single stranded joining reaction near the left end creates a single strand circle within a branched figure-8 (F-8). Replication or repair convert F-8 into a double stranded mini-circle in which the abutted ends are separated by a single base pair. In the second step, (iv), two concerted single strand nicks at the junction of the two ends (the mini-circle junction) linearize the element for insertion into the target. Recent evidence has indicated that the minicircle junction (mj) is the site of a new hybrid promoter which, it has been hypothesized, drives the 2nd step of transposition. Two hypotheses form the basis of this study to explore further the mechanistic details of this novel pathway. (a) the minicircle is either a mandatory intermediate in the pathway or it is by product of a linear excision product, and its mj promoter is essential for transposition, or it is an artifact. (b) different protein-DNA interactions are required for the single strand cleavage in step 1 and the two concerted single strand cleavages in step 2. Three specific objectives will drive this project. (i) to examine the role of the minicircle junction and its promoter in the transposition pathway. (ii) to explore the basis for the asymmetry of the 1st step cleavage reaction, and (iii) to decipher the attributes which determine that, a single strand nick will occur in the 1st step and two concerted single strand nicks will occur in the second step.
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