MECHANISTIC STUDIES OF A TRANSPOSITIONAL PATHWAY IN IS2

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DESCRIPTION: To date, two classical paradigms have described transpositional mechanisms. The cut and paste mechanism describes the simple insertion process for elements like the retroviruses and Tn7/Tn10. A more complex process, which requires replication of the element and results in cointegrate formation, has been described for elements such as phage Mu. Very recently, a third transpositional pathway has been hypothesized for two small insertion sequences, IS2 and IS911. The hypothesis for IS2 involves a novel 2-step process which is initiated by an asymmetric one-ended cleavage and joining reaction in which the right end is joined to the left (target) end to form a transposon minicircle with two abutted ends. In the second step, it is proposed that the minicircle is inserted into the target site by a two-ended concerted event which follows cleavage of the abutted end. Both of these steps should be catalyzed by the transposase, the principal protein encoded by these insertion sequences. This study proposes to test this hypothesis in IS2: (I) by determining how the transposase binds to and cleaves the new sequence created by the junction of the IS2 ends in minicircles; (ii) by determining whether the transposase interacts with the left end differently when it is a target in the one-ended reaction than when it is a cleavage substrate in the two-ended reaction; and (iii) by testing the hypothesis that a junction promoter formed by the abutted IS2 ends in the minicircle is needed to provide the elevated levels of transposase required for insertion events.