探討酵母菌中去氧核糖核酸酶影響TERRA調控端粒重組之機制

Abstract

端粒位於真核生物染色體末端區域，其功能對於保護染色體及維持基因成分相當重要。然而隨著細胞分裂次數增加，端粒會不斷地縮短，若不修復的話細胞會走入老化的階段(senescence)。端粒長度除了可以透過端粒酶調控之外，亦可藉由ALT (alternative lengthening of terlomeres)的途徑來維持，此機制是建立在同源重組的基礎上將端粒末端序列延長。在端粒酶缺失的酵母菌中可觀察到有一群細胞利用端粒重組(telomere recombination)的方式度過老化存活下來，他們可以透過端粒前方區域Y’ element (type I)或是末端的TG1-3片段(type II)來進行重組，使端粒長度得以維持。其中，Type II重組與人類的ALT癌細胞較為相似，然而目前對於端粒重組的機制仍不清楚。在我們先前的研究發現一種稱為TERRA的telomeric long-noncoding RNA會參與在端粒重組的過程中，TERRA會和端粒DNA形成RNA:DNA hybrid (R-loop)，且在端粒酶缺失的酵母菌中TERRA會促使端粒重組的發生，因此我們想近一步探討TERRA到底是如何引發端粒重組。由於TERRA與端粒所形成之R-loop是一個很特殊的構造，有機會被細胞中具結構特異性之去氧核醣核酸酶(DNase)進行辨認並切割，進而調控端粒的重組。我利用基因剔除的技術在端粒酶缺失的酵母菌系統中發現DNA break repair nuclease Pso2、nucleotide excision repair nuclease Rad10和base-excicsion repair相關的核酸酶，包括了DNA N-glycosylases/AP lyases (Ntg1, Ntg2和 Ogg1), AP endonucleases (Apn1, Apn2) 以及DNA deadenylase Hnt3，這些基因對於端粒重組的發生影響不大。然而有趣的是，我們發現Holliday junction reslovase YEN1及MUS81可能在type I和type II survivor形成過程中扮演調控的角色，並且又以type II重組路徑中顯得更為重要。

Telomeres are the terminal regions of eukaryotic linear chromosomes and they are essential for the maintenance of chromosome stability and genome integrity. After several replication events, telomeres will get shortened and cells can enter senescence. Telomere length could be maintained by telomerase or a recombination-based mechanism termed ALT (alternative lengthening of telomeres). In yeast Saccharomyces cerevisiae lacking telomerase, two types of recombination were used to maintain telomeres. Elongation of telomeres could be mediated through recombination between subtelomeric Y’ elements (Type I) or telomeric TG1-3 tracks (Type II), but these two recombination pathways are mediated through genetically-distinct mechanisms. The type II recombination pathway is similar to that observed in human ALT cells. Previously we found that a non-coding telomere transcript, TERRA, forms RNA:DNA hybrids to facilitate type II telomere recombination. However, the mechanism of how TERRA affects recombination is unclear. Since the RNA:DNA hybrid formed by TERRA is likely to form a R-loop structure, nuclease(s) that recognize and process the R loop structure might also function in telomeres. Here I use genetics approach to identify and test the role of nucleases that involved in telomere recombination. I found deletion of DNA break repair nuclease Pso2, nucleotide excision repair nuclease Rad10, and several nucleases involved in base-excision repair including, DNA N-glycosylases/AP lyases (Ntg1, Ntg2 and Ogg1), AP endonucleases (Apn1, Apn2) and DNA deadenylase Hnt3 did not affect type II recombination. Interestingly, deletion of YEN1 and/or MUS81, genes encoding two known Holliday junction resolvases, did not have an effect on telomere recombination but these nucleases might be involved in survivors formation regulation, and showed more significant effect on type II pathway.