PHYSICAL CHEMISTRY OF RECOMBINATIONAL INTERMEDIATES

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Recombination is a fundamental phenomenon that generates genetic diversity in living organisms. It is the long-range goal of that work to obtain a detailed understanding of the DNA chemistry which characterizes this process. The information sought will afford molecular-scale data on the rearrangement of genetic material in living organisms. Recombination occurs in meiosis, where gametes are produced, in the development of antibodies, in the generation of trypanosome variable surface antigens, and in the insertion of viral DNA Into host genomes. Both generalized and site-specific recombination proceed through four-stranded intermediates, known as Holiday junctions. Stabilized Holiday junctions appear to form 2-told symmetric complexes from four strands of DNA; two strands of DNA are helical, while the other two strands cross over between helical domains. The response of this structure to temperature and divalent cations will be determined. An isomer of this structure in which the strands reverse helical and crossover roles exists: this isomerization will be characterized, using hydroxyl radical as a structural probe. Branch-point migration is another isomerization reaction; it is responsible for the instability of junction. This isomerization will be studied by NMR. The dependence upon substrate of the resolution of junctions by T4 Endonuclease VII will be studied. Junctions will be crystallized for crystallographic study. The thermodynamics of junction formation will be determined from calorimetric experiments. Knots, a related recombination-specific unusual topology of DNA, will be made from synthetic oligodeoxynucleotides; these will be characterized as well. This series of experiments will yield information which will provide detailed understanding about recombination in the development and evolution of organisms.