The mechanism that causes genomic instability in nondividing aging cells is unknown. Our previous study of mutant yeast suggested that 2 types of replication-independent endogenous DNA double-strand breaks (RIND-EDSBs) exist and that they play opposing roles. The first type, known as physiologic RIND-EDSBs, were ubiquitous in the G0 phase of both yeast and human cells in certain genomic locations and may act as epigenetic markers. Low RIND-EDSB levels were found in mutants that lacked chromatin-condensing proteins, such as the high-mobility group box (HMGB) proteins and Sir2. The second type is referred to as pathologic RIND-EDSBs. High pathological RIND-EDSB levels were found in DSB repair mutants. Under normal physiologic conditions, these excess RIND-EDSBs are repaired in much the same way as DNA lesions. Here, chronological aging in yeast reduced physiological RIND-EDSBs and cell viability. A strong correlation was observed between the reduction in RIND-EDSBs and viability in aging yeast cells ( r = 0.94, P < 0.0001). We used galactose-inducible HO endonuclease (HO) and nhp6a∆, an HMGB protein mutant, to evaluate the consequences of reduced physiological RIND-EDSB levels. The HO-induced cells exhibited a sustained reduction in RIND-EDSBs at various levels for several days. Interestingly, we found that lower physiologic RIND-EDSB levels resulted in decreased cell viability ( r = 0.69, P < 0.0001). Treatment with caffeine, a DSB repair inhibitor, increased pathological RIND-EDSBs, which were distinguished from physiologic RIND-EDSBs by their lack of sequences prior to DSB in untreated cells [odds ratio (OR) ≤1]. Caffeine treatment in both the HO-induced and nhp6a∆ cells markedly increased OR ≤1 breaks. Therefore, physiological RIND-EDSBs play an epigenetic role in preventing pathological RIND-EDSBs, a type of DNA damage. In summary, the reduction of physiological RIND-EDSB level is a genomic instability mechanism in chronologically aging cells.-Thongsroy, J., Patchsung, M., Pongpanich, M., Settayanon, S., Mutirangura, A. Reduction in replication-independent endogenous DNA double-strand breaks promotes genomic instability during chronological aging in yeast.