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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29GM054630-05
Application #
6180994
Study Section
Special Emphasis Panel (ZRG3-BBCA (01))
Program Officer
Lewis, Catherine D
Project Start
1996-09-30
Project End
2001-03-31
Budget Start
2000-09-01
Budget End
2001-03-31
Support Year
5
Fiscal Year
2000
Total Cost
$23,554
Indirect Cost
Name
Research Institute of Fox Chase Cancer Center
Department
Type
DUNS #
064367329
City
Philadelphia
State
PA
Country
United States
Zip Code
19111
Feng, Jin-an (2007) Improving pairwise sequence alignment between distantly related proteins. Methods Mol Biol 395:255-68
Li, Wei; Wen, Liting; Feng, Jin-An (2006) RepairNET: a bioinformatics toolbox for functional exploration of DNA damage response. J Cell Physiol 207:293-9
Wen, Liting; Li, Wei; Sobel, Marc et al. (2006) Computational exploration of the activated pathways associated with DNA damage response in breast cancer. Proteins 65:103-10
Wang, Junwen; Feng, Jin-An (2005) NdPASA: a novel pairwise protein sequence alignment algorithm that incorporates neighbor-dependent amino acid propensities. Proteins 58:628-37
Wen, Liting; Feng, Jin-An (2004) Repair-FunMap: a functional database of proteins of the DNA repair systems. Bioinformatics 20:2135-7
Wang, Junwen; Feng, Jin-An (2003) Exploring the sequence patterns in the alpha-helices of proteins. Protein Eng 16:799-807
Crasto, C J; Feng, J (2001) Sequence codes for extended conformation: a neighbor-dependent sequence analysis of loops in proteins. Proteins 42:399-413
Crasto, C J; Feng, J A (2000) LINKER: a program to generate linker sequences for fusion proteins. Protein Eng 13:309-12
Tang, L; Li, J; Katz, D S et al. (2000) Determining the DNA bending angle induced by non-specific high mobility group-1 (HMG-1) proteins: a novel method. Biochemistry 39:3052-60
Feng, J A; Crasto, C J; Matsumoto, Y (1998) Deoxyribose phosphate excision by the N-terminal domain of the polymerase beta: the mechanism revisited. Biochemistry 37:9605-11