PHYSICAL CHEMISTRY OF RECOMBINATIONAL INTERMEDIATES

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Genetic recombination occurs universally in living organisms, from viruses to humans. The basic feature of recombination is the interaction of two pieces of DNA to yield new genetic material that may physically incorporate segments of both interacting molecules. The consequences of these changes include viral integration in species ranging from bacteriophages in prokaryotes to retroviruses, such as HIV, in humans. Genetic transposition, gene duplications with clinical consequences, the development of the immune system, and the diversification of human gene pools through meiotic crossovers all result from recombination processes. Recombination is not just a passive phenomenon: Recently, it has become a tool used extensively by geneticists to modify the genes of experimental species. The goals of this work are to understand the structure, dynamics and thermodynamics of DNA molecules involved in genetic recombination. DNA model systems that can be designed and synthesized in the laboratory are emphasized. Key among these molecules is the Holliday junction, which is a four-stranded branched recombination intermediate. It has been modeled recently by asymmetric analog molecules, which have been found to have asymmetric two-domain antiparallel structures; whereas this finding implies asymmetric genetic products as a function of sequence at the branch, it will be determined whether molecules with natural symmetry also have asymmetric domain structures. The parallel or antiparallel nature of the intermediate as a substrate for resolution enzymes will also be established, using catenated molecules. Holliday junctions undergo two isomerizations with genetic consequences, switching between domain structures and migration of the branch point; the products of these isomerizations are directly related to the genetic results of recombination. Catenated and knotted versions of the Holliday intermediate will be used to explore and control, these isomerizations. Attempts will be made to determine the three-dimensional structure of the intermediate by crystallography. Knotted DNA molecules are also recombination intermediates. It is possible to construct model synthetic DNA knots, whose physical properties will be determined. Knots contain fixed node-structures, which will be used to determine the nodal specificities of DNA topoisomerases. In addition, their topological properties enable modeling the natural, torsionally-stressed state of DNA. These unusual DNA structures will be used to seek cellular activities that recognize them uniquely, or for which they are cognate substrates. The research to be performed will answer key questions about the DNA molecules that are involved in recombination. It will yield concrete structural and physical knowledge about these unusual DNA molecules. The ultimate goal is to provide molecular control over natural recombination phenomena and over recombinational interventions done today in the laboratory and tomorrow in the clinic.