The Ku subunit of telomerase binds Sir4 to recruit telomerase to lengthen telomeres in S. cerevisiae.
The Ku subunit of telomerase binds Sir4 to recruit telomerase to lengthen telomeres in S. cerevisiae.
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DOI：
10.7554/elife.07750
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发表时间：
2015-07-28
期刊：
影响因子：
7.7
通讯作者：
Zappulla DC
中科院分区：
文献类型：
--
作者：
Hass EP;Zappulla DC
在酿酒酵母和人类中，端粒酶RNA亚基与Ku结合，Ku是一种环状蛋白异二聚体，以其DNA修复功能而闻名。Ku结合酵母端粒酶RNA促进端粒延长和端粒酶募集到端粒，但这是如何实现的仍然未知。利用端粒长度分析和染色质免疫沉淀，我们发现Sir4——一种先前鉴定的ku结合蛋白，是端粒沉默染色质的一个组成部分——是ku介导的端粒延长和端粒酶募集所必需的。我们还发现，特异性地将Sir4直接拴在ku结合缺陷的端粒酶RNA上，可以将原本缩短的端粒恢复到野生型长度。这些发现表明Sir4是ku介导的端粒酶募集的端粒结合靶点，并为Sir4竞争的Rif1和Rif2蛋白如何负性调节酵母端粒长度提供了一种机制。在细胞核内，DNA被包装成称为染色体的结构。每条染色体的末端都被称为端粒的重复DNA序列所覆盖，端粒保护染色体免受损害。每当细胞分裂时，端粒就会缩短。如果端粒长度低于临界水平，细胞就会死亡或进入不能再分裂的状态。在细胞分裂过程中，一种叫做端粒酶的酶通常会将端粒恢复到原来的长度。端粒酶由几种蛋白质和一个RNA分子组成。在酵母和人类中，一种叫做Ku的蛋白质是端粒酶的一部分。Ku与端粒酶的RNA亚基结合，帮助酶找到端粒并与端粒相互作用。先前的研究表明，Ku无法单独将端粒酶募集到染色体上。一种名为Sir4的蛋白质与端粒结合，缺乏Sir4的细胞端粒较短，但其背后的原因尚不清楚。Hass和Zappulla用生化方法证实了Ku与Sir4结合的先前报道。另外的实验提供了遗传证据，表明这种结合相互作用对端粒酶适当延长端粒很重要。端粒酶的RNA亚基不能有效地与Ku结合的细胞具有短端粒。Hass和Zappulla直接将Sir4连接到这个有缺陷的RNA上，并发现这将缩短的端粒恢复到正常长度，这表明Sir4通常与Ku结合以募集端粒酶。发现这种招募模式也有助于解释另外两种端粒蛋白（Rif1和2）如何限制端粒延长；它们与Ku-Sir4的招募竞争，形成长度调节系统。综上所述，Hass和Zappulla的研究结果提供了强有力的证据，证明Sir4与Ku协同控制染色体末端的延长。未来的研究有望揭示酵母中端粒酶控制系统的精确空间和时间要求。此外，由于Ku已被报道为人类端粒酶的一个亚基，未来的研究还可以探索人类细胞是否使用类似的策略。DOI: http://dx.doi.org/10.7554/eLife.07750.002
In Saccharomyces cerevisiae and in humans, the telomerase RNA subunit is bound by Ku, a ring-shaped protein heterodimer best known for its function in DNA repair. Ku binding to yeast telomerase RNA promotes telomere lengthening and telomerase recruitment to telomeres, but how this is achieved remains unknown. Using telomere-length analysis and chromatin immunoprecipitation, we show that Sir4 – a previously identified Ku-binding protein that is a component of telomeric silent chromatin – is required for Ku-mediated telomere lengthening and telomerase recruitment. We also find that specifically tethering Sir4 directly to Ku-binding-defective telomerase RNA restores otherwise-shortened telomeres to wild-type length. These findings suggest that Sir4 is the telomere-bound target of Ku-mediated telomerase recruitment and provide one mechanism for how the Sir4-competing Rif1 and Rif2 proteins negatively regulate telomere length in yeast. DOI: http://dx.doi.org/10.7554/eLife.07750.001 Inside a cell's nucleus, DNA is packaged into structures called chromosomes. The ends of every chromosome are capped by repeating sequences of DNA known as telomeres, which protect the chromosomes from damage. Every time a cell divides, the telomeres shorten. If telomere length falls below a critical level, the cell can die or enter a state in which it can no longer divide. During cell division, an enzyme called telomerase normally restores telomeres to their original length. Telomerase is made up of several proteins and an RNA molecule. In yeast and humans, a protein called Ku is one part of the telomerase enzyme. Ku binds to the RNA subunit of telomerase and helps the enzyme find and interact with the telomeres. Previous research has shown that Ku is unable to work alone to recruit telomerase to the chromosome. A protein called Sir4 binds to telomeres and cells lacking it have short telomeres, but the reason behind this was not known. Hass and Zappulla confirmed previous reports that Ku binds to Sir4 using a biochemical approach. Additional experiments provided genetic evidence that this binding interaction is important for telomerase to lengthen telomeres appropriately. Cells in which the RNA subunit of telomerase is unable to bind effectively to Ku have short telomeres. Hass and Zappulla directly tethered Sir4 to this defective RNA and found this restored the shortened telomeres to a normal length, indicating that Sir4 normally binds Ku to recruit telomerase. Discovering this mode of recruitment also helps to explain how two other telomeric proteins (Rif1 and 2) limit telomere lengthening; they compete with Ku-Sir4 recruitment to form a length-regulating system. Taken together, Hass and Zappulla's results provide strong evidence that Sir4 cooperates with Ku to control the lengthening of chromosome ends. Future research will hopefully reveal the precise space and time requirements for this telomerase-controlling system in yeast. Additionally, because Ku has been reported to be a subunit of human telomerase, future studies could also explore whether human cells use a similar strategy. DOI: http://dx.doi.org/10.7554/eLife.07750.002