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.
| Nickoloff, J A; Singer, J D; Heffron, F (1990) In vivo analysis of the Saccharomyces cerevisiae HO nuclease recognition site by site-directed mutagenesis. Mol Cell Biol 10:1174-9 |
| Nickoloff, J A; Singer, J D; Hoekstra, M F et al. (1989) Double-strand breaks stimulate alternative mechanisms of recombination repair. J Mol Biol 207:527-41 |
| Nickoloff, J A; Chen, E Y; Heffron, F (1986) A 24-base-pair DNA sequence from the MAT locus stimulates intergenic recombination in yeast. Proc Natl Acad Sci U S A 83:7831-5 |
