Bacteriophage Mu Transposition and the Role of Gyrase Binding Sites

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Pato 9727991 The bacterial virus Mu, by virtue of its being both a virus and a transposon, is unusual among transposons in its large size and its high frequency of transposition. Amplification of the 37.2 kb Mu DNA during the lytic cycle occurs by a series of replicative transposition events which take place within the bacterial nucleoid, despite the constraints imposed by the complex structure of that body. Concerns about potential problems with synapsing the ends of prophage DNA within the nucleoid, an early step in the transposition process, led to studies which uncovered a strong gyrase binding site (SGS) in the center of the Mu genome that is required for efficient replication. An extensive search was undertaken to find chromosomal or plasmid sites which could replace the SGS in Mu replication. No other sites have been found, focusing attention on what is unusual about the SGS. This project focuses on two important questions raised by the model: 1) How does the Mu site promote synapsis of the Mu prophage ends and 2) Why is a novel mechanism required for promoting the synapsis? The first question will be addressed by determining what is special or unique about the Mu site. A detailed genetic and biochemical analysis of the SGS and of other strong gyrase binding sites is being performed with the goal of defining the elements of the SGS that are responsible for its ability to function in Mu DNA replication. The second question is being addressed by exploring the factors, such as the binding of transposase molecules to the ends of a Mu prophage, that are responsible for the long delay in Mu replication in the absence of the SGS. These studies are designed to provide insights into the mechanism of viral DNA replication and the importance of DNA structure in biological function. Of particular interest is the role of DNA topology in the modulation of function.