Telomere capping in non-dividing yeast cells requires Yku and Rap1.

The assembly of a protective cap onto the telomeres of eukaryotic chromosomes suppresses genomic instability through inhibition of DNA repair activities that normally process accidental DNA breaks. We show here that the essential Cdc13-Stn1-Ten1 complex is entirely dispensable for telomere protection in non-dividing cells. However, Yku and Rap1 become crucially important for this function in these cells. After inactivation of Yku70 in G1-arrested cells, moderate but significant telomere degradation occurs. As the activity of cyclin-dependent kinases (CDK) promotes degradation, these results suggest that Yku stabilizes G1 telomeres by blocking the access of CDK1-independent nucleases to telomeres. The results indeed show that both Exo1 and the Mre11/Rad50/Xrs2 complex are required for telomeric resection after Yku loss in non-dividing cells. Unexpectedly, both asynchronously growing and quiescent G0 cells lacking Rap1 display readily detectable telomere degradation, suggesting an earlier unanticipated function for this protein in suppression of nuclease activities at telomeres. Together, our results show a high flexibility of the telomeric cap and suggest that distinct configurations may provide for efficient capping in dividing versus non-dividing cells.

The cell division cycle puts up with unprotected telomeres: cell cycle regulated telomere uncapping as a means to achieve telomere homeostasis.

Telomeres have unique properties that distinguish natural chromosomal ends from accidental DNA double-strand interruptions arising elsewhere in the genome. However, the slightest perturbation in their unique organization may obliterate this distinction, channelling chromosomal ends into unwarranted repair events, eventually causing genome instability. Recent results revealed that the processing of both dysfunctional telomeres and accidental DNA double strand breaks (DSB) by DNA repair activities is tightly regulated in a cell cycle-dependent manner by the S phase-promoting cell cycle kinase CDK1 (Clb-Cdc28p). Surprisingly, the cell cycle determinants and the timing of processing at unprotected telomeres closely match the requirements of other transactions that occur at telomeres. In particular, the replenishment of telomeric repeats by telomerase is tightly linked to cell cycle progression and occurs in the same interval. Furthermore, cell survival in the absence of essential telomeric proteins being dependent on telomere-telomere recombination mechanisms may require a similar regulation. Thus, a temporally limited state of telomere dysfunction leading to chromosome end processing may represent a well-governed cell cycle event that constitutes an integral part of the assembly of a new functional telomere.

DNA degradation at unprotected telomeres in yeast is regulated by the CDK1 (Cdc28/Clb) cell-cycle kinase.

In the absence of functional telomeric cap protection, the ends of eukaryotic chromosomes are subject to DNA damage responses that lead to cell-cycle arrest and, eventually, genomic instability. However, the controlling activities responsible for the initiation of genome instability on unprotected telomeres remained unclear. Here we show that in budding yeast, unprotected telomeres undergo a tightly cell-cycle-regulated DNA degradation. Ablation of the function of essential capping proteins Cdc13p or Stn1p only caused telomere degradation in G2/M, but not in G1 of the cell cycle. Accordingly, G1-arrested cells with unprotected telomeres remained viable, while G2/M-arrested cells failed to recover. The data also show that completion of S phase and the activity of the S-Cdk1 kinase were required for telomere degradation. These results strongly suggest that after a loss of the telomere capping function, telomere-led genome instability is caused by tightly regulated cellular DNA repair attempts.

Mechanism of early biphasic activation of poly(ADP-ribose) polymerase-1 in response to ultraviolet B radiation.

The damage to DNA caused by ultraviolet B radiation (280-320 nm) contributes significantly to development of sunlight-induced skin cancers. The susceptibility of mice to ultraviolet B-induced skin carcinogenesis is increased by an inhibitor of the DNA damage-activated nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP), hence PARP activation is likely to be associated with cellular responses that suppress carcinogenesis. To understand the role of activated PARP in these cellular functions, we need to first clearly identify the cause of PARP activation in ultraviolet B-irradiated cells. Ultraviolet B, like ultraviolet C, causes direct DNA damage of cyclobutane pyrimidine dimer and 6, 4-photoproduct types, which are subjected to the nucleotide excision repair. Moreover, ultraviolet B also causes oxidative DNA damage, which is subjected to base excision repair. To identify which of these two types of DNA damage activates PARP, we examined mechanism of early PARP activation in mouse fibroblasts exposed to ultraviolet B and C radiations. The ultraviolet B-irradiated cells rapidly activated PARP in two distinct phases, initially within the first 5 minutes and later between 60-120 minutes, whereas ultraviolet C-irradiated cells showed only the immediate PARP activation. Using antioxidants, local irradiation, chromatin immunoprecipitation and in vitro PARP assays, we identified that ultraviolet radiation-induced direct DNA damage, such as thymine dimers, cause the initial PARP activation, whereas ultraviolet B-induced oxidative damage cause the second PARP activation. Our results suggest that cells can selectively activate PARP for participation in different cellular responses associated with different DNA lesions.