9420804 Pato Bacteriophage Mu/ by nature 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 Mu DNA during the lytic cycle occurs by a series of replicative transposition events which take place within the bacterial nucleoid, despite the restraints 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 in the center of the Mu genome which is required for efficient replication. An hypothesis was proposed that the gyrase site is involved in organizing the topology of the supercoiled prophage DNA to assist in synapsis of the Mu ends. Several predictions of the model were tested during the previous grant period and the results to date offer strong support for the hypothesis. The present research continues the studies with Mu and extends them to include studies on the possible role of strong gyrase binding sites in chromosomal structure. The prediction that the Mu strong gyrase site can assist in bringing together interacting DNA sequences that are equidistant from the gyrase site will be tested/ and features of gyrase and of the binding site that are required for this activity will be examined. A powerful selection procedure for the isolation and characterization of potential strong gyrase binding sites from the E. coli chromosome is presented. Fragments of chromosomal DNA will be cloned into the center of a prophage deleted for its gyrase site, and hence unable to form a plaque; any fragment that restores plaque forming ability will be a candidate for one carrying a strong gyrase site. %%% Studies of bacterial and viral replication in this research have the potential to provide information useful in interfering with virus growth in other organisms. *** ??
