Title: Characterization of the Saccharomyces cerevisiae sirtuin family and their roles in lifespan regulation
Description: The budding yeast Saccharomyces cerevisiae has proven to be a valuable model organism in the field of aging research. S. cerevisiae responds to calorie restriction, a treatment that has been shown to increase the lifespan of almost every organism tested. Calorie restriction (CR) consists of reducing the caloric intake of an organism without provoking malnutrition. Sir2, originally found in yeast, is the founding member of a family of NAD + -dependent deacetylases, collectively known as sirtuins, which are highly conserved across species. In yeast, it extends replicative lifespan by promoting the formation of compact heterochromatin through histone deacetylation at the ribosomal DNA (rDNA) locus. This prevents the excision of extrachromosomal rDNA circles (ERCs), which accumulate in the mother cell and eventually cause its death. In S. cerevisiae , CR activates Sir2, the formation of ERCs is suppressed, and lifespan is extended. Deletion of SIR2 prevents calorie restriction from extending lifespan in yeast. Overexpression of the Sir2 homologs in C. elegans and D. melanogaster increases the lifespan of these organisms. Sirt1, the mammalian homolog of Sir2, is involved in the regulation of many pro-survival pathways.

The deacetylation reaction catalyzed by Sir2 releases O-acetyl-ADP-ribose and nicotinamide. Nicotinamide is a very potent inhibitor of Sir2. The nicotinamidase PNC1 is upregulated during conditions of mild stress that extend lifespan, thus relieving the inhibitory effects of nicotinamide on Sir2 activity. Whether Sir2 was regulated by an increase in NAD + levels during CR or by a decrease in nicotinamide was widely debated. In this dissertation I describe an in vivo reporter system that shows that NAD + levels do not rise during CR and other conditions that extend lifespan. This suggests that clearance of nicotinamide by Pnc1 regulates Sir2 activity during CR. In a screen designed to find factors that extend lifespan, I found that a Sir2 homolog, Hst2, is able to increase S. cerevisiae replicative lifespan through the same mechanism as Sir2. This prompted me to study the role of all yeast Sir2 homologs ( HST1, HST2, HST3, HST4 ) in the regulation of replicative and chronological lifespan, two different measures of yeast aging. I show that all yeast sirtuins are able to increase lifespan, and mechanisms by which they achieve this. These findings could set the framework for future studies on sirtuins in higher organisms.