Project Summary DNA damage presents a threat to genomic integrity and if unrepaired can contribute to apoptosis or disease-causing mutations. Thus, accurate repair of lesions is necessary and requires a team of repair enzymes. Topoisomerases are a class of DNA repair enzymes that relieve torsional stress during a variety of DNA metabolic processes, such as replication and repair. Type IA topoisomerases are best characterized for their decatenase activity, which in yeast acts in concert with Rmi1 downstream of RecQ helicases. It has also been shown that eukaryotic topoisomerase 3 (Top3; a type 1A topoisomerase) cleaves RNA molecules, but it is unclear whether Top3 also possesses the genomic ribonucleotide excision function seen with topoisomerase 1. In this proposal, we outline research aimed at understanding the substrate requirements of yeast Top3 that promote efficient targeting and enzymatic activity. Further, we plan to investigate the requirement for other proteins, such as the RecQ helicase and Rmi1, in mediating Top3 substrate specificity. Because errors in DNA repair can contribute to development of diseases, such as cancer, knowledge gained from the proposed studies can provide insight into the role of type 1A topoisomerases in both normal and erroneous genomic maintenance. Additionally, a number of cancer-associated mutations in the human type 1A topoisomerase, TopoIIIa, have been collected. Thus, results from these studies could contribute to a greater understanding of the role of type 1A topoisomerases in tumorigenesis.