Age-dependent effects of SIR2 dosage on yeast gene expression and replication fork stalling

Michelle Yuen
Pomona College

In the yeast Saccharomyces cerevisiae, mother cells age one generation each time they bud to produce a daughter cell, while daughter cells begin at age 0. The average life span of a cell is 25-30 generations, however this can be altered by mutations, some of which affect the ribosomal DNA (rDNA). Sir2p functions as a NAD+-dependent histone deacetylase (HDAC) to silence transcription of the telomeric, rDNA, and silent mating type genes (Tanny et al., 1999). Deletion of SIR2 results in a 30% shorter life span, while moderate overexpression extends life span 30% (Kaeberlein et al., 1999). Previous studies suggest that yeast aging may be caused by stalled replication forks and subsequent recombination events (Hoopes et al., 2002), including the formation of extrachromosomal rDNA circles. In this model, SIR2 extends life span by suppressing replication initiation at the rDNA, resulting in fewer stalled replication forks (Pasero et al., 2002). An alternative aging hypothesis is that there is a genetic program of aging that is regulated by Sir2p through its HDAC activity. I am using microarrays to test whether there are age-dependent changes in gene expression that are regulated by Sir2p, which would indicate support for a genetic program of aging. I will also be using 2-dimensional gel electrophoresis to confirm the results found by Pasero et al. and also examine if overexpression of SIR2 further diminishes the frequency of stalled forks, which would support a role for fork stalling in aging.