THE ROLE OF NUCLEASES IN RECOMBINATION IN YEAST

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.