MECHANISM OF INSERTION SEQUENCE TRANSLOCATION

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The study of two different types of specialized recombination is proposed. Transpositional recombination, the integration of transposable DNA into a target site, will be investigated using two contrasting transposons as model systems. These are IS903, which generally transposes by a donor-destructive, non-replicative pathway, and GammaDelta which uses a replicative (cointegrate) pathway. Site specific recombination, the recombination between two specific sites by a reciprocal, conservative breakage reunion reaction, will be studied using the resolution of GammaDelta cointegrates by the transposon encoded TnpR protein, resolvase. The following specific projects are proposed. I Transpositional Recombination. 1) Purification and characterization of the transposase proteins of IS903 and GammaDelta, with transposition in vitro as the ultimate goal. 2) Analysis of the interaction of transposon ends and transposase (a) by mutational analysis of the transposon terminal inverted repeats and (b) by identification of DNA binding domains within the transposase protein by isolation of mutants of transposase that show altered sequence specificity. 3) Further analysis of the mechanism of deletion formation by IS903 and IS10. II Site-specific Recombination. 1) Mutational dissection of the cointegrate resolution process, (a) by developing assays for the various steps in recombination and (b)\by isolating and characterizing mutants specifically defective in each step. 2) Detailed analysis of the molecular interactions between (a) the N-terminal domain of resolvase and the crossover site, and (b) the C-terminal domain and the resolvase-binding DNA segment. 3) Analysis of the apparent resolvase-induced bending of res DNA.