Our goal is to understand the mechanism underlying the recognition and repair of DNA damage. We will use yeast as outlined in the specific aims: (1) The Rad52 DNA repair protein: We will continue our molecular genetic characterization of the Rad52 DNA repair pathway using newly developed Rad52-GFP fusions. We will continue to explore Rad52 functional domains and analyze the pathways defined by novel gamma-ray sensitive, hyper- recombination rad52 alleles. We will also explore the post translational modification that produces multiple Rad52 protein species. (2) Sml1, a negative regulator of RNF: We will investigate the regulatory circuitry of the Rnr inhibitor, Sml1. We will determine which pathways and what modifications regulate Sml1 degradation during S phase and after DNA damage. We will investigate the role of other proteins in Sml1 phosphorylation and/or regulation and develop a new tool to allow the visualization of the DNA damage response in living cells. (3) The Top3/Sgs1 DNA topoisomerase/helicase complex: We will continue to explore the genetic and biochemical interactions between Top3 and Sgs1. We will isolate and identify new alleles of SGS1 that separate top3 suppression from its DNA damage sensitivity. To further define the TOP3 function, we will attempt to isolate non-sgs1 suppressors of top3 as well as suppressors of catalytically inactive Top3. We will investigate those Top3 functions that do not require Sgs1. We will further characterize SHU1, a suppressor of sgs 1 hydroxyurea sensitivity. We will also explore the cell biology and biochemistry of this important protein complex. These combined genetic, biochemical and cell biological approaches to the many issues related to the recognition and repair of DNA damage in yeast will continue to yield new insights into this important biological process.