Transposons are ubiquitous DNA elements that vary widely in size and complexity. The smallest and simplest transposons are insertion sequences only a few hundred base pairs long. Larger complex members of the family include eucaryotic RNA tumor viruses (AIDS-related viruses) and procaryotic DNA viruses. A transposons alters chromosome function by moving from site to site in the genome of its host. Impressive advances made in understanding the biochemical mechanics of several specific transposons stand in stark contrast to ignorance about regulatory mechanisms that constrain or activate transposons in genetic populations. One of the most efficient transposons is a large DNA virus--phage Mu--that is capable of transposing 100 times per hour. Our general goal is to understand how Mu organizes its large DNA domain during transposition reactions and how the Mu domain interacts with domains in the host bacterium. DNA domains define a limit for DNA supercoiling and they confine certain classes of DNA-DNA site interactions. The topoisomerases are intimately involved in domain formation in both procaryotes and eucaryotes. There are two general goals in this application. One is to explore the interwound supercoil structure of the bacteriophage Mu domain. This will be carried out by creating a set of modified vires carrying pairs of the gamma-delta transposon's resolution (res) sites. A gyrase anchored at the center of Mu is critical for transposition in vivo. We will test the influence of this single gyrase enzyme on the supercoiled structure of the Mu domain in vivo by measuring its effect on recombination rates of res sites spotted at different points in the Mu genome. The second goal is to compare the supercoil domain structure of phage Mu with a similar sized segment of the bacterial genome. We intend to learn how a Mu domain interacts with a bacterial chromatin during transposition. The bacterial domain we will study is the region from minute 41 to 42 in Salmonella typhimurium, which starts at the his operon and includes two operons that are transcribed under anaerobic growth conditions, pdu and cob. We plan to answer two questions. First, how do the changes in chromatin structure that occur when cells change from aerobic growth to anaerobic growth affect Mu transposition? This study will exploit a new technique called Muprinting, which reveals the detailed integration pattern of Mu as it moves from site to site. The differences between the patterns in aerobic and anaerobic cultures give clues to the location and activities of proteins bound to DNA under both conditions. Second, we will introduce gamma-delta res sites into this region of the bacterial genome and compare the site specific recombination rates of gamma-delta sites in this domain to the data set from Mu.
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