Our long term objective is to understand the molecular interaction of DNA with proteins which would influence the function and metabolism of nucleic acids. As first step towards achieving this goal we plan to determine the 3 dimensional structures of the individual proteins and the macromolecular complexes which are involved in DNA replication, recombination and repair. Subsequently we will relate the structural data to genetic, biochemical and biophysical information in order to advance our understanding of the molecular mechanism of these biolobical events. This type of detailed knowledge will provide profound insights into a variety of important research areas such as cancer, aging and genetic engineering. In this proposal, we plan to use electron microscopy to determine the 3 dimensional structures of gp32*I, a T4 DNA helix destabilizing protein at 7Angstrom resolution, of RecA protein, an E. coli recombination enzyme and of RecA and DNA complex at low resolution (greater than 25 Angstrom). We will preserve these specimens in a frozen, hydrated state, image them under low dose, low temperature conditions to reduce radiation damage and use computer processing methods to reconstruct the 3 dimensional density maps. We will develop a number of image processing procedures needed to achieve the specified resolution in the reconstructions. The work on gp32*I protein will represent the first example of a high resolution structural study on the crystal of a soluble protein by electron microscopy. The methodology resulting from this project will have broad applicability to other thin protein crystals. At 7 Angstrom we expect to identify the secondary structure of the protein. This will provide answers to some of the questions about structure-function relationships in the protein. Moreover this structural data will form the basis of future high resolution study of the protein by electron or x-ray diffraction analysis. We will attempt to grow large crystals of gp32 and of gp32*I for atomic structure determination by X-ray diffraction method. The low resolution study of RecA and its complex with DNA in a filamentous form will provide information about the size and shape of the protein and its complex with DNA. These will enable us to evaluate current models of RecA-DNA interaction. Because this proposal involves a considerable effort to develop novel methodology in electron microscopy, a 5 year support is requested to complete the proposed tasks.
| Morgan, D G; Grant, R A; Chiu, W et al. (1992) Patch averaging of electron images of GP3*I crystals with variable thickness. J Struct Biol 108:245-56 |
| Grant, R A; Schmid, M F; Chiu, W (1991) Analysis of symmetry and three-dimensional reconstruction of thin gp32*I crystals. J Mol Biol 217:551-62 |
| Chang, C F; Rankert, D A; Jeng, T W et al. (1988) Cryo electron microscopy of unstained, unfixed RecA-cssDNA complexes. J Ultrastruct Mol Struct Res 100:166-72 |
| Prasad, B V; Chiu, W (1987) Sequence comparison of single-stranded DNA binding proteins and its structural implications. J Mol Biol 193:579-84 |
| Grant, R A; Schmid, M F; Chiu, W et al. (1986) Alignment and merging of electron microscope images of frozen hydrated crystals of the T4 DNA helix destabilizing protein gp32*I. Biophys J 49:251-8 |
| Chiu, W (1986) Electron microscopy of frozen, hydrated biological specimens. Annu Rev Biophys Biophys Chem 15:237-57 |
