THE ROLE OF RIND-EDSBs IN CHRONOLOGICAL AGING YEAST CELL

Abstract

Recently, we discovered a new class of endogenous DNA double strand breaks (EDSBs). They are found in all cell types and phases. Because these EDSBs are found in G0, we named them replication independent EDSBs (RIND-EDSBs). In human, RIND-EDSBs are retained in hypermethylated DNA within deacetylated chromatin but without H2AX. RIND-EDSB repair is more precise than pathologic EDSB repair. While methylated RIND-EDSBs are repaired by precise ATM mediated non-homologous end joining (NHEJ) during G0, radiation induced DSBs are repaired by fast and more error prone DNA-PKcs mediated NHEJ pathway. We evaluated RIND-EDSBs in chronological aging yeast. Not only the viability but also RIND-EDSBs are decreased in aged yeasts. This study was divided into three main parts. First is to set up the method for measure RIND-EDSBs in Saccharomyces cerevisiae. Second is to prove that RIND-EDSBs are biological breaks that have the molecular mechanisms regulation in retained RIND-EDSBs. Finally, the relationship between viability and RIND-EDSBs in yeast was evaluated. We found that the RIND-EDSB levels increased significantly in yeast strains lacking proteins involved in some histone deacetylases, endonucleases, topoisomerase, and DNA repair regulators. In contrast, RIND-EDSB levels were downregulated in the mutants that lack chromatin-condensing proteins, such as the high-mobility group box proteins, and Sir2. Moreover, RIND-EDSB occurrences have specificity in terms of sequence pattern, that is, the majority of breaks occurred right after the sequence “CGK”. The specificity in “CGK” sequence is not due to chance as “CGK” sequence in yeast genome occurs non-primarily. To evaluate the roles of RIND-EDSBs, we induced global EDSB repair by HO induction. We found that yeasts at 3 days after HO induction had lower RIND-EDSB levels. The lower RIND-EDSB levels were associated with lowering viability, increasing caffeine sensitivity and increasing fast repaired non-CGK RIND-EDSBs. Therefore, we concluded that, the genomic levels of RIND-EDSBs are evolutionally conserved, dynamically regulated, non-randomly presented and may be influenced by genome topology, chromatin structure, and the efficiency of DNA repair systems. Furthermore, we found two classes of RIND-EDSBs. The first occurs non-randomly, preceding the break by CGK sequence. The others do not possess specific sequence. Finally, reduction of RIND-EDSBs during chronological aging led to increased production of pathologic fast repair non-CGK RIND-EDSBs and consequently reduce cell viability. These evidences suggested that the CGK-RIND-EDSBs may be epigenetic marks, playing important role in preventing genomic instability and cellular aging. Cells may normally maintain a certain level of RIND‐EDSBs, a reduction of which may affect cell viability.