A long term goal of this project is to understand the assembly, control, and function of nucleoprotein complexes that promote Hin-catalyzed site-specific DNA inversion. Work during the past funding period established the overall structure of the tetrameric catalytic core of the Hin synaptic complex, provided strong evidence that recombination of DNA strands was mediated by rotation of Hin subunit pairs within the tetramer, and showed how the Fis/enhancer system together with DNA supercoiling controls subunit rotation. Future work will entail an ensemble of genetic, biochemical, and structural approaches and will emphasize the mechanism by which the Fis/enhancer element activates initial reaction steps as well as controls the subunit rotation process. Mechanistic analysis will commence on an enzyme from a different class of serine recombinase, emphasizing its novel properties. A prominent theme of the project concerns the DNA binding properties and regulatory roles of nucleoid proteins on recombination and transcription reactions. New aspect on the control of phage lambda site specific recombination by nucleoid proteins will be pursued. X- ray crystal structure analyses of complexes bound by the nucleoid protein Fis will be extended to higher- order complexes which contain the phage lambda Xis protein and the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD). These structural studies will provide new information on the role of DNA conformational flexibility on indirect recognition and cooperative DNA binding by proteins. Cellular Fis levels vary enormously with respect to growth phase and growth rates, being the most abundant DNA binding protein in E. coli under rapid growth conditions but virtually absent in stationary phase. Mechanisms of transcriptional regulation by Fis through specific and non-specific DNA binding will be investigated. Recent in vitro evidence suggests Fis may play an important role in modulating chromosome structure because of its DNA-bending and looping activities. Fis effects on chromosome structure will be addressed by a combination of in vivo and in vitro approaches, including collaborative single-DNA molecule approaches as well as by bulk-phase in vitro and in vivo experiments.
This research aims to understand the mechanism and control of specialized DNA recombination reactions, particulariy those of the less well understood serine recombinase family. A broader goal is to understand how abundant non-histone DNA binding and bending proteins modulate chromosome structure and control DNA recombination and gene expression.
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