Many cellular processes are controlled by a precise interplay between multiple factors. We are just beginning to understand at the molecular level how multi-component nucleoprotein complexes are assembled and catalytically activated. The Hin site-specific DNA inversion reaction is well suited for investigating these issues. In this reaction, three DNA segments and a minimum of 2 Hin recombinase dimers and two Fis enhancer protein dimers assemble into an invertasome structure in a reaction that requires DNA supercoiling. Invertasome assembly is also assisted by either the prokaryotic HU or members of the eukaryotic HMG1/2 family of non- specific DNA binding proteins that promote DNA looping. A primary aim of this project is to elucidate the molecular structure of the catalytically competent invertasome. Structural information is available for each of the components and the geometric configuration of DNA strands within the recombination complex is known. A series of experiments are proposed which are guided, in part, by computer modeling to elucidate the molecular structure of the invertasome and how it is catalytically activated by Fis plus the enhancer DNA segment. In addition, the mechanism by which the DNA strands are exchanged within the invertasome will be investigated. These studies will advance our understanding of the process and control of specialized DNA recombination reactions, which are found in many biological contexts. Aberrant recombination events can lead to serious chromosomal aberrations and cancer. The functions of non-histone DNA binding proteins in diverse reactions is becoming increasingly recognized as an important level of control. As an example, the Fis protein not only regulates Hin- catalyzed DNA inversion, but also controls transcription of different promoters and stimulates viral excision from the chromosome, another site-specific DNA recombination reaction. The mechanisms by which Fis activates and represses transcription and functions in lambda excision will be investigated, particularly to determine the relevant roles of Fis-mediated DNA bending versus direct contact with the appropriate target protein. The distribution of high affinity sites versus non-specific binding at different regions of the Escherichia coli genome will also be explored to provide information regarding the role of Fis in controlling growth phase and growth rate gene expression and in mediating overall nucleoid structure.
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