• Heffron, Fred (PI)

    Project: Research project

    Project Details


    Of the site specific nucleases reported in eukaryotes only HO has
    been characterized in detail. The enzyme has been purified, its
    gene has been identified and cloned, it recognition site
    identified, and the product of cleavage determined. Most
    important, the biological role of the enzyme has been identified:
    it is required for mating type interconversion in Saccharomyces
    cerevisiae because it initiates a specific recombination event by
    making a double-strand break (DSB). The aim of this proposal is
    to continue our work on this project. HO is such a highly
    specific nuclease that it makes a single DSB in the entire yeast
    genome. The DSB generated by cleavage at its recognition site
    will be used to make specific breaks in chromosomes and study
    the genetic consequences of the break. In addition, the
    enzymology of the nuclease will be investigated in detail. The
    kinetic and thermodynamic parameters of the enzyme will be
    determined and possible intermediates in the reaction will be
    examined and possible intermediates in the reaction will be
    examined to determine their role in recombination if any. Very
    little is known about nucleases in any eukaryotic organism.
    Several human hereditary diseases appear to result from
    decreased activity of nucleolytic repair enzymes. Patients with
    these defects show increased incidance of malignant cancer.
    Yeast is the ideal organism for a study of nucleases because of
    the ease of genetic manipulation and the availability of large
    quantities of cells for biochemical studies. The long term goal
    of this research is to identify and characterize the important
    nucleases from this eukaryotic organism.
    Effective start/end date7/1/846/30/93


    • National Institutes of Health


    • Medicine(all)
    • Biochemistry, Genetics and Molecular Biology(all)


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