Transposons are ubiquitous DNA elements that have the ability to move from one genomic location to another. Their movement can cause mutations and genome rearrangements and plays an important role in genome evolution including the horizontal transfer of genetic information between species. Many bacterial transposons encode resistance to antibiotics and their mobility is primarily responsible for the acquisition of multiple-drug resistance by medically important bacteria. The study of bacterial transposons has allowed them to be developed as powerful molecular tools and to serve as paradigms for genome rearrangements that occur in higher eukaryotes; the processes of retroviral integration an immunoglobulin rearrangement are directly related to that of bacterial transposition. The long term objective of this research is to determine the molecular mechanism of transposition of the bacterial insertion sequence IS903 and to understand how this process is regulated. This will be achieved by developing a structure-function analysis of the IS-encoded transposase protein, which will allow the role of transposase and the DNA sites it recognizes during transposition to be characterized. A multidisciplinary approach using genetics, biochemistry and biophysical studies will be used to accomplish these goals. The complementary nature of this approach will facilitate the functional characterization of the IS903 transposase while extending our understanding of the biology of transposons and the genome rearrangements they cause.
The aims are: 1. To investigate the novel target preferences of IS903. Key features of the preferred target site will be determined by analyzing the use of a series of different DNA substrates. Targeting to specific regions of the chromosome will be monitored in a variety of genetic backgrounds to determine host factors that influence genome targeting. 2. To isolate mutants of transposase that have acquired novel properties. A variety of genetic screens and selections have been developed to facilitate these analyses. In particular, mutant transposases will be sought that have altered binding specificity, increased transposition activity, and the ability to transpose replicatively. 3. To establish biochemical assays that will identify specific transposase-DNA contacts, define stable domains of the protein and facilitate the development of an in vitro transposition system.
