PHYSICAL CHEMISTRY OF RECOMBINATIONAL INTERMEDIATES

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DESCRIPTION: Genetic recombination occurs universally in living organisms, from viruses to humans. Much of DNA metabolism is devoted to ensuring the stability necessary for its role as genetic material, but recombination affords the flexibility necessary for species to adapt to changing environments. Its key feature is the interaction of two pieces of DNA to yield new genetic material that may incorporate segments of both interacting molecules. The consequences of recombination include viral integration in species ranging from prokaryotic bacteriophages to retroviruses, such as HIV, in humans. Genetic transposition, the development of the immune system, and the diversification of human gene pools all result from recombination processes. Recombination is not just a passive phenomenon; it is a tool used 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. Dr. Seeman focuses on DNA model systems that can be designed and synthesized in the laboratory. Key among these molecules is the Holliday junction, which is a four-stranded branched intermediate, but Dr. Seeman often uses molecules with two junctions to obtain conformational purity; these parallel double crossover molecules are meiotic intermediates. Two isomerizations with genetic consequences are associated with Holliday junctions, branch migration and crossover isomerization. Dr. Seeman will ask three questions about branch migration: (1) Are all migratory positions equally likely? (2) Do antiparallel Holliday junctions branch migrate? (3) Do parallel double crossover molecules branch migrate: He will ask two questions about crossover isomerization: (1) Is it really a physical process? (2) Can Holliday junctions braid, which is related to its mechanism in molecules with parallel domains? He also seek to establish the relationship between the site of the branch point and sequences required for cleavage of Holliday junctions by RuvC resolvase. Dr. Seeman has discovered a new DNA motif that he suggests may support the search for homology in homologous recombination. He will challenge this suggestion with experiments to characterize its structure and its interactions with RecA. 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.