This project entails the characterization of DNA molecules involved in the processes of DNA recombination. These are typically branched molecules, whose 3D structures are only partially known. A key issue is the recognition of homology by double stranded DNA molecules. We have established that there is an unusual DNA structure that is formed by homologous molecules when they are in a supercoiled plasmid.
Specific Aim 1. The structure that is formed is a dumbbell-like, where the shaft contains the homologous DNA. We plan to ascertain the structure of the shaft of the homology structure. To see if (as hypothesized) the structure is indeed PX-DNA, we will proceed v^ath chemically-based experiments, including crosslinking the strands together andpadlocking the circle. We would like to have the best physical evidence that we can get on the shaft. The next level of characterization will entail electron microscopic examination of dumbbells that have been derivatized by metallic nanoparticles that will be visible. We will use this labeling scheme to try to make the relevant portions of the dumbbell molecules visible to the cryo-EM, so that those methods can be applied to this key structure.
Specific Aim 2. We have produced crystals of a variety of branched and otherwise unusual DNA species relevant to recombination. These include designed rhombohedral lattices that self-assemble to form macroscopic crystals. We plan to determine the structures of these lattices. We also have crystals of the inside of a double crossover molecule (related to meiotic intermediates) that diffract to atomic resolution, and a third crystal that contains an unusual linkage. We will determine the structures ofthese molecules and will establish their 3D structures. In addition, we will try to improve the resolution of some of the larger self-assembling lattices by a lashing technique that will, if successful, lead to the ability to insert guests into the lattice.
The presence of homology is central to the key biological processes of genetic recombination and DNA repair. DNA in the cell is capable of assuming many different structures in response to conditions. We have discovered that in the presence of homology supercoiled DNA (which is the state of DNA in the cell) forrns a special structure as a consequence of the homology;we have shown that the structure can be captured by crosslinking within the cell. We aim to characterize this structure to the greatest extent possible.
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