We have isolated a hyper-recombination mutants, TOP3 that identifies a novel eukaryotic topoisomerase in the yeast, Saccharomyces cerevisiae. The predicted open reading frame of the TOP3 gene is homologous to the bacterial type, I topoisomerase, TopA, but not homologous to eukaryotic type I or II enzymes. We plan to investigate the biochemical properties of this novel enzyme and to define its substrate preferences. Null mutations exhibit pleiotropic phenotypes such as slow growth, reduced diploid formation and a sporulation defect. We will investigate the consequences of these pleiotropies by examining mitotic and meiotic nuclear division, spindle formation and the mating reaction. Genetic experiments will address these pleiotropies by in vitro mutagenesis of the TOP3 gene and by searching for genetic suppressors of these phenotypes. A slow growth suppressor, sgs1, has already been isolated and will be cloned and mapped using a modified Ty mutagenesis method that we will develop. Lastly, we will begin to investigate whether the TOP3 gene is a general feature of all cell by attempting to identify a homologue in the evolutionarily distant yeast Schizosaccharomyces pombe by using either nucleic acid homology or protein homology as a probe. It is our goal to understand the role that this novel topoisomerase plays in cells growth and development. The process of genetic recombination is observed in all living organisms. Although this process has important biological consequences both for individuals and for populations, and the details of its mechanism has been under intense study in a number of laboratories for years, we still do not have a good understanding of how this process helps living organisms survive. During his previous investigations into the mechanism of recombination in yeast, the author of this proposal discovered anew gene involved in this process; this gene encoded an enzyme of a type not previously seen in yeast or higher organisms. Mutants defective in this gene also had defects in many aspects of their life cycle. This is a proposal to investigate how this gene is involved with these other life processes.
