Hydrogen peroxide (H 2 O 2 ), a reactive oxygen species (ROS), is known to induce oxidative stress and apoptosis. U937 cells treated with H 2 O 2 were shown to produce high molecular weight (HMW) DNA fragments ∼50 to 100 kb in size in <1 min. The formation of these HMW DNA fragments is reversible and shown to be mediated by DNA topoisomerase II (TOP2). Following this initial event, formation of irreversible HMW DNA fragments and nucleosomal ladders occurs. Our results thus demonstrate a potential role of TOP2 in oxidative damage of DNA and apoptotic cell death.

Selenium (Se) compounds, which are the most extensively studied cancer chemopreventive agents, induce apoptotic death of tumor cells. In the current study, we show that selenite-induced apoptosis involves DNA damage. We showed that selenite-

Inactivation of the budding yeast telomere binding protein Cdc13 results in abnormal telomeres (exposed long G-strands) and activation of the DNA damage checkpoint. In the current study, we show that inactivation of Cdc13p induces apoptotic signals in yeast, as evidenced by caspase activation, increased reactive oxygen species production, and flipping of phosphatidylserine in the cytoplasmic membrane. These apoptotic signals were suppressed in a mitochondrial ( o ) mutant. Moreover, mitochondrial proteins (e.g. MTCO3) were identified as multicopy suppressors of cdc13-1, suggesting the involvement of mitochondrial functions in telomere-initiated apoptotic signaling. These telomereinitiated apoptotic signals were also shown to depend on MEC1, but not TEL1, and were antagonized by MRE11. Our results are consistent with a model in which single-stranded G-tails in the cdc13-1 mutant trigger MEC1-dependent apoptotic signaling in yeast.Cdc13p is a budding yeast telomere binding protein (1-3). Cdc13p appears to be a multifunctional protein involved in both telomere replication and protection (1-4). Its role in telomere replication has been suggested based on the findings that it interacts with the catalytic subunit of DNA polymerase ␣ and an essential subunit of telomerase, Est1p (4), and a mutation form of CDC13, cdc13-2, shows the telomerase negative phenotype (3). Its essential role in protecting yeast telomeres is presumably related to its binding to the exposed G-strand (1-3, 5). Cdc13-1p (P371S), a temperature-sensitive mutant form of Cdc13p, is unable to protect telomeres at the restrictive temperature (1). At the non-permissive temperature, cells with the cdc13-1 allele exhibit extensive degradation of telomeres from the 5Ј-ends, the C-rich strands (C-strands). The C-strand degradation results in long single-stranded Gtails (up to 20 kb) at the 3Ј-ends. The abnormal telomeres in cdc13-1 at the non-permissive temperature lead to RAD9-dependent cell cycle arrest at the G 2 /M phase, as well as cell death (1, 6). However, the mechanism of cell death triggered by Cdc13p inactivation is unclear. It was assumed that essential gene deletion because of the C-strand degradation is the cause of the cell death.Yeast apoptosis is not well understood. Bax, Bcl-2, and other established apoptotic proteins have not been found in yeast cells. However, expression of human BAX in yeast induces cell death (7,8), accompanied by mitochondrial alkalinization and cytosol acidification (9). This process can be blocked by coexpression of human anti-apoptosis protein Bcl-2 (8). Additionally, aged mother cells and H 2 O 2 -treated cells have shown apoptosis markers in yeast, including flipping of phosphatidylserine (PS) 1 from the inner leaflet to the outer leaflet of the cytoplasmic membrane, DNA damage (by TUNEL assay), and reactive oxygen species (ROS) induction (10, 11). More recently, a caspase-like protease Yca1p has been identified in yeast and was found to regulate yeast cell death induced by H 2 O 2 treatment (12). Moreover, a bro...

Single-stranded DNA has been speculated to be the initial signal in the DNA damage signaling pathway. We showed that introduction of single-stranded DNA with diverse sequences into mammalian cells induced DNA damage as well as apoptosis signals. Like DNA damaging agents, single-stranded DNA up-regulated p53 and activated the nuclear kinase ataxia telangiectasia mutant (ATM) as evidenced by phosphorylation of histone 2AX, an endogenous ATM substrate. Single-stranded DNA also triggered apoptosis as evidenced by the formation of caspase-dependent chromosomal DNA strand breaks, cytochrome c release, and increase in reactive oxygen species production. Moreover, single-stranded DNA-induced apoptosis was reduced significantly in p53 null cells and in cells treated with ATM small interfering RNA. These results suggest that single-stranded DNA may act upstream of ATM/p53 in DNA damage signaling.