縮短端粒的酵母菌修復雙股DNA斷裂之缺失

Abstract

端粒(telomere)的存在能夠維持染色體的穩定性，而端粒的複製則仰賴端粒酵素的活性。缺乏端粒酵素活性的哺乳類癌細胞或酵母菌細胞可藉由ALT (Alternative Lengthening of Telomeres)的重組方式來維持端粒長度。本研究發現由缺乏端粒酵素的酵母菌細胞衍生而來的type I 存活者對於DNA的傷害有高度敏感性。藉由比較野生型和typeI存活者之突變率的實驗，我們認為此現象並不是由於基因的改變而發生。另一方面，利用端粒PCR及TRF (Telomere Restriction Fragment)分析法來追蹤缺乏端粒酵素的酵母菌從孢子形成到存活者出現的過程，我們發現端粒的長度和存活者的敏感度有密切的關係。此外本實驗也發現type I 對於雙股DNA斷裂的敏感性必須仰賴MEC1基因的存在，顯示此現象可能來自於檢查點持續地被啟動及其所造成的延滯的細胞週期。先前的研究指出在缺乏端粒酵素的酵母菌中，縮短且失去正常功能的端粒會引發相似於DNA損傷的反應。為了進一步釐清此現象是否因為縮短的端粒將細胞中的修復蛋白質凝聚至染色體末端而造成染色體其他位置的斷裂無法有效被修復，我們利用染色質免疫沉澱法來觀察同源性重組機制中的Rad51蛋白質在野生型和type I存活者中與單一DSB (double-strand break)結合的能力。結果顯示此能力在type I存活者相較於野生型，確實有下降的現象。

Telomere maintenance is required for chromosome stability, and telomeres are typically replicated by the action of telomereas. In both mammalian tumor and yeast cells that lack telomerase, telomeres are maintained by an alternative (ALT) recombination mechanism. Here we demonstrated that the budding yeast Saccharomyces cerevisiae type I survivors derived from telomerase-minus cells were hypersensitive to DNA-damaging agents. Tests on mutation rates showed no difference between type I survivors and WT cells, indicating that no specific genetic alterations might be responsible for the sensitive phenotype. On the other hand, telomere PCR and spot assays which tracked the sensitivity of telomerase-minus cells from spore to survivors suggested a correlation between telomere length and bleomycin sensitivity. We further discovered that this sensitivity is MEC1 dependent and probably results from the persistent checkpoint activation and prolonged cell cycle arrest. Previous studies have reported that short dysfunctional telomeres can induce a DNA damage response in S. cerevisiae in the absence of telomerase. Our analyses of the HR protein (Rad51p) binding efficiency at DSB sites by quantitative chromatin IP suggest that the sensitive phenotype might correlate with less efficient recruitment of repair proteins to the bona-fide DSB induced by exogenoues DNA-damaging agents when eroded telomeres give signal of break sites as well.