Functional studies of the top 1-binding protein topors

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DESCRIPTION (provided by applicant): Topoisomerase I is the target of an important class of anti-cancer drugs, the camptothecins. The overall goal of this project is to more completely understand the functions of topoisomerase I in normal and malignant cells, with the hope of eventually improving cancer treatment. Towards this end, the proposal focuses on topors, a protein that binds topoisomerase I and p53. Current results indicate that topors functions as both an E3-type ubiquitin and SUMO ligase and stimulates the sumoylation of topoisomerase I and p53 in vitro. In addition, topors dynamically associates with promyelocytic leukemia nuclear bodies, which are implicated in cellular sumoylation processes and in carcinogenesis. Protein and mRNA expression studies indicate that topors is widely expressed in normal human tissues, but not in cancer cell lines. In contrast to normal colon tissue, topors protein expression is frequently undetectable in human colon cancers. These findings will be pursued using biochemical and genetic approaches. The first aim includes detailed mapping of topors domains involved in ubiquitin and SUMO conjugation processes. In addition, the effects of topors-induced sumoylation on topoisomerase I will be studied in vitro and in cells In the second aim, mechanisms underlying the association of topors with promyelocytic leukemia nuclear bodies will be explored, including analysis of binding of the promyelocytic leukemia protein by topors, and determination of whether topors is sumoylated in cells. The third aim focuses on investigation of the differential expression of topors in normal versus malignant human tissues. In addition to further characterization of the loss of topors expression in malignancies, Ioss-of-heterozygosity and mutation of the topors gene will be investigated, as well as mechanisms underlying the anti-proliferative effects of topors in cell lines. These studies will be complemented by investigation of the effects of loss of topors function using transgenic mice in Aim 4. Potential roles for topors in the suppression of tumorigenesis and in cellular sensitivity to camptothecin will be investigated. The proposed experiments should significantly increase current knowledge regarding the function of topors and may lead to new strategies in the prevention and treatment of cancer