MECHANISTIC ANALYSIS OF DNA TRANSPOSITION BY PHAGE MU

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Transpositional recombination is a powerful generator of genetic diversity. A disparate set of elements, isolated from many different organisms, promote their own movement from one DNA site to another by transposition. The long term goal of this project is to understand the molecular mechanism of transposition by phage Mu. The extraordinary efficiency of Mu transposition makes it an ideal system for mechanistic studies. The DNA cleavage and joining reactions central to transposition are known. Mu, the bacterial element Tn10, and retroviruses use essentially the same DNA cleavage and joining steps, and each encodes a protein that promotes these reactions. Recently, it has become clear that the active form of the Mu transposase is a tetramer of the protein bound to the paired ends of the element. This insight provides an opportunity to analyze how the multiple DNA cleavage and joining reactions are coordinated within the tetrameric protein complex. By using partially defective transposases and site specific DNA-protein crosslinking, the functional organization of this active complex will be probed. These experiments, along with additional crosslinking, chemical modification, and mutagenesis studies, will also locate and dissect the active sites of the Mu transposase. Genetic and biochemical experiments to address the role of the Mu transposition proteins, and specific host factors, in recruiting the cellular replication machinery to the transposition intermediate are also proposed. The relative simplicity of the reaction, the stability of the protein-DNA complexes, and the requirement for multiple DNA sites during complex assembly, also make Mu transposition an ideal system to study the mechanisms underlying assembly of protein-DNA complexes. Understanding of the protein-DNA assembly process in Mu transposition is likely to provide insights generalizable to the fields of transcription and replication control. A biochemical description of transposition should also facilitate development of transposon-mediated mutagenesis in organisms where this is not yet feasible. In addition, the impact of transpositional recombination on human health is immense. The rapid horizontal spread of antibiotic resistance is largely a result of transposable elements moving, on broad host range plasmids and as conjugative transposons, throughout bacterial populations. Furthermore retroviruses, including HIV, integrate into the host chromosome by a mechanism identical to Mu transposition. Understanding the molecular mechanism of this important process should assist the future design or discovery of agents that may prevent these undesirable consequences of transposition.