The observation that Sir2 protein levels decline precipitously 20 hours before rDNA recombination rates increase significantly led us to further examine the relationship between Sir2 depletion and age-associated LOH. We tested the effect of SIR2 gene copy number on rDNA recombination using diploid strains homozygous for a tandem repeat of SIR2 under control of its native promoter (SIR2OE). Previous studies indicated that this modest level of over-expression is sufficient to increase silencing of RNA Polymerase II-dependent transcription within the rDNA array [34], [37] and extend RLS [32], while higher expression of Sir2 is toxic [38]. Sir2 over-expression in young (log-phase) diploid cells was verified by Western blot (Figure 6A). The decline in Sir2 protein levels was partially suppressed in the SIR2OE strain: Sir2 levels in SIR2OE populations were elevated at 26 hours compared to wild type, but declined precipitously to wild type levels by 50 hours (Figure 6A).
