In order to examine this issue, we found it necessary to further develop methods employed to examine ERC levels with age. Previous analyses of ERC levels have been technically limited to the examination of relatively young cells (∼10 generations) [42], whereas we observe age-associated increases in LOH after the median life span of our diploid strains (36 generations for UCC5185) [16]. To better understand the kinetics of ERC accumulation, we purified aged mother cells from MEP cultures and quantified ERC levels by Southern blotting. To improve ERC quantitation, we removed the linear rDNA array by digestion with RecBCD exonuclease, a highly processive exonuclease that does not degrade intact or nicked circular DNA [43] (Figure S2). Monomeric, dimeric, and multimeric ERC species were identified based on their migration rates [44] (Figure 8A) and quantified by densitometry (Figure 8B).
