MECHANISTIC ANALYSIS OF DNA TRANSPOSITION BY PHAGE MU

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Phage Mu transposes at an extraordinarily high frequency and many fundamental studies of transposition have been done using Mu. It is now clear that key aspects of this recombination mechanism are shared by Mu and many other transposable elements that invade prokaryotic and eukaryotic organisms. The long term goal of this project is to understand the molecular mechanism of Mu transposition. It has recently become clear that Mu transposase is a member of a structurally related family of proteins that includes many transposases and the retroviral integrases. These proteins catalyze recombination as multimeric complexes bound to DNA. Understanding of the structural organization of a transposase-DNA complex in most advanced for the Mu transposase. The active form of Mu transposase is a homotetramer bound simultaneously to the three segments of DNA that participate in recombination. The specific goals of this proposal are aimed at providing a thorough description of how the DNA molecules are arranged within this tetrameric protein complex and how the complex is assembled. We will use protein- DNA and protein-protein cross-linking to map the regions of Mu transposase that interact with the DNA at the recombination sites and provide the protein-protein surfaces that hold the tetramer together. Assembly of the active transposase tetramer absolutely depends on binding of the protein to specific DNA sites; the structural basis of this DNA-dependence will be investigated using multiple probes for changes in protein conformation. Genetic and biochemical experiments to address how an appropriate DNA target site is selected during recombination are also proposed. This analysis of the mechanisms underlying the assembly and activation of the protein-DNA complexes involved in transposition is likely to provide insights into the mechanisms that control transcription and replication as well. The impact of transposition on human health is immense. The rapid spread of antibiotic resistance genes is largely a result of transposable elements moving throughout bacterial populations. Furthermore, retroviruses, including HIV, integrate into the host chromosome via a mechanism nearly identical to transposition. A related recombination reaction is also responsible for assembly of the immunoglobulin and T-cell receptor genes during development of the vertebrate immune system. Understanding the molecular mechanism of this important class of genetic recombination should assist the future design or discovery of agents that may prevent the undesirable consequences of transposition.