UNDERSTANDING THE ROLE OF THE RAD12 HELICASE IN S POMBE

My broad long term objectives are to understand how the cancer prone disease Bloom's syndrome occurs. The gene defect in Bloom's syndrom (BLM) has been cloned and characterized and homologs in several model systems identified. Specifically, I will study the function of the rad12 the BLM homolog in S. pombe. My specific aims are to establish its role as a reverse gyrase when it functions in conjunction with topoisomerase III. In addition I want to definitively show that rad12 protein acts as a transcription factor and identify genes that it regulates. I will express rad12 and topoIII in yeast expression systems and then develop an assay that will allow me to demonstrate that these proteins function together to act as a reverse gyrase (i.e. they add positive supercoils to relaxed covalently closed circular DNA. I will also use both microarrays and DNA affinity binding to identify genes whose expression is rad12 dependent. Ultimately I will establish the phenotype of cells lacking genes identified in this screen.