Our goal is to understand the mechanisms underlying the recognition and repair of DMA damage. We use yeast as an experimental organism to continue our studies on three different aspects of this essential biological process 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 Rad52-fluorescent protein fusions. We will follow up on leads from our genomic screen of the yeast gene disruption library to study the role of nuclear pore proteins and other DNA metabolism genes in Rad52 focus formation. We will continue to explore Rad52 functional domains and analyze the pathways defined by novel gamma-ray sensitive, hyperrecombination rad52 alleles. We will also explore the post-translational modification that produces multiple Rad52 protein species. (2) Sml1, a negative regulator of RNR: We will continue to 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 using Sml1-fluorescent protein fusion to visualize 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, Sgs1 and a newly discovered interacting protein, Nce4. We will study non-sgs1 suppressors of top3. We will further characterize the role of the Shu suppressor complex (Shu1/Shu2/Psy3/Csm2) in error-free repair. We will screen the yeast gene disruption library for additional genes that interact with Top3, Sgs1 and Nce4 using a novel method that we recently developed. These combined genetic 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. ? ?
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