Intellectual Merit The advent of new DNA sequencing technologies and a wealth of sequenced genomes have intensified the need to develop innovative approaches for extracting functional insights from these data. Although homology is a useful principle, homologous proteins sometimes have different functions across species, and these differences can reconfigure regulatory pathways governing how organisms respond to environmental changes. Consequently, extrapolating function from model species can be misleading. To understand how proteins acquire new functions and the biological consequences of these changes, this project focuses on a group of proteins named Sir2 deacetylases, which are found in all kingdoms of life. These enzymes are proposed to coordinate life-cycle transitions with nutrient availability. Importantly, Sir2 deacetylases display varied modes of action and may enable species-specific responses to nutritional conditions. This project employs a combination of genome-scale and classical functional studies to investigate the evolutionary history of Sir2 in genetically tractable yeast species. The first part of the project examines how Sir2 orthologs have evolved new functions by acquiring interactions with a variety of protein complexes that repress gene expression, and the second part explores the consequences of such changes as they relate to the physiology of cells. To elucidate the steps by which Sir2 acquired its function in Saccharomyces cerevisiae, the functions of Sir2 orthologs will be compared in species at appropriate evolutionary distances from S. cerevisiae, namely in Candida lusitaniae, a representative species of the Candida clade, and in the yeast Kluyveromyces lactis. As these species have different responses to nutrient levels in terms of specific aspects of their physiology, this project has the potential to elucidate the roles of Sir2 in such responses. Broader Impact This project will provide training opportunities for two graduate students, several Duke undergraduate students conducting independent studies, and summer students participating in two programs designed to encourage disadvantaged students to pursue careers in science. The Duke Summer Research Opportunity Program (SROP) targets undergraduates, and the North Carolina Section Project SEED program supports high school students. In addition, this project will promote the recruitment and retention of female scientists by supporting the careers of a graduate student and the principal investigator, who are both women. Finally, the development of additional yeast species, such as K. lactis and C. lusitaniae, as model organisms will greatly facilitate future studies in molecular evolution by enabling researchers to test their hypotheses using multiple experimentally tractable yeast species that are separated by hundreds of millions of years of evolution.