The long-term goals of this project are to understand: (a) the mechanism of Hin mediated site-specific DNA recombination, (b) specific DNA sequence recognition by Hin and Fis proteins, and (c) protein-DNA interactions by a non-specific DNA binding protein, NHP6a. These three functions constitute some of the most basic biological processes. The knowledge learned from this study will help us to understand more complex systems of similar functions. Site-specific recombination plays a major part in normal cell development, and aberrant rearrangements can lead to cancer and birth defects. The proposed work is concerned with understanding the molecular mechanism of a model site-specific recombination involving an inversion of 1 kb segment of DNA in Salmonella. This reaction controls the alternate expression of two flagella genes enabling the bacteria to evade from host cell attack. It requires the presence of three proteins: Hin, Fis, and HU (which can be substituted by NHP6a, a yeast protein). Biochemical data have suggested that initiation of the reaction requires two recombination sites bound with Hin dimers paired together in association with an enhancer bound with Fis proteins, forming a nucleoprotein complex, called invertasome. Our approach to elucidate the reaction mechanism at atomic level is to study the structures of the components of the reaction and their complexes with DNA. Dr. Feng will use x-ray crystallographic techniques to determine the structures of intact Hin recombinase and its complex with the recombination site. Dr. Feng will study Fis/DNA complexes and determine what is the specificity of its DNA binding, how it is able to recognize multiple sequences of DNA, and what DNA distortion it induces and how these structural changes relate to its function in the inversion reaction. Dr. Feng will study the protein-DNA interactions by NHP6a. To ensure formation of a stable complex for crystallization, Dr. Feng propose to construct a Hin-NHP6a fusion protein, where the specific binding of Hin will guide the NHP6a onto the DNA with limited degree of freedom. This study will also allow Dr. Feng to test the idea of designing a DNA recognition molecule which covers extended length of DNA sequence. The structures of these complexes will serve as basic building blocks for construction of a realistic model for an invertasome, which will help us to understand the mechanism of the reaction.
