Ku must load directly onto the chromosome end in order to mediate its telomeric functions.
Ku must load directly onto the chromosome end in order to mediate its telomeric functions.
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DOI：
10.1371/journal.pgen.1002233
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发表时间：
2011-08
期刊：
影响因子：
4.5
通讯作者：
Bertuch AA
中科院分区：
文献类型：
--
作者：
Lopez CR;Ribes-Zamora A;Indiviglio SM;Williams CL;Haricharan S;Bertuch AA
Ku异二聚体与酿酒酵母端粒相关，在那里它影响端粒结构和功能的几个方面。虽然Ku通过预先形成的通道与DNA末端结合，但其通过这种机制与端粒结合的能力可能受到已知直接结合染色体末端的因素的挑战。这导致了不确定性，Ku本身是否直接结合到端粒末端，以及末端缔合是否对Ku的端粒功能至关重要。为了解决这些问题，我们构建了DNA末端结合缺陷Ku异二聚体通过改变氨基酸残基Ku 70和Ku 80，预计接触DNA。这些突变体继续与它们已知的端粒相关的伙伴相关联，例如端粒沉默所需的因子Sir 4和端粒酶的RNA组分TLC 1。尽管有这些相互作用，我们发现Ku突变体与端粒染色质和端粒末端保护，长度调节和沉默功能的空样缺陷的关联显着减少。与Ku缺失菌株相反，DNA末端结合缺陷Ku突变体导致诱导双链断裂时不精确的末端连接熟练度增加，而不是显着降低。这一结果进一步支持了Ku端粒末端结合的特异性丧失导致端粒缺陷，而不是Ku功能的整体丧失。在这些突变体中观察到的广泛的端粒缺陷使我们提出Ku是终端端粒帽的一个组成部分，在那里它促进了一个特定的架构，是端粒功能和维护的核心。端粒帽调节端粒复制，并防止天然染色体末端被加工为DNA双链断裂（DSB）。在包括芽殖酵母在内的多个物种中，存在一幅关于构成端粒帽的因子以及它们如何与端粒DNA相关联的详细图片。Ku是一种保守的异源二聚体，参与端粒生物学和DSB修复的多个方面，但在何处放置Ku尚不清楚。尽管Ku能与DNA末端结合，但其进入端粒末端可能受到端粒结合蛋白和/或高阶端粒结构的限制。Ku也可能通过其端粒相关的结合伴侣被募集到端粒。在这里，我们解决是否Ku负载直接到端粒末端，是否直接DNA结合是至关重要的端粒功能。使用结构指导的诱变，我们产生了末端结合缺陷的酵母Ku异源二聚体，保留了与Ku的已知端粒结合伙伴的能力。这些末端结合缺陷的异源二聚体表现出端粒协会的显着减少，并有缺陷的所有Ku的端粒功能。我们的研究结果表明，Ku确实是端粒帽的一个组成部分，其加载到端粒末端是至关重要的端粒功能，也许，一个特定的端粒结构。
The Ku heterodimer associates with the Saccharomyces cerevisiae telomere, where it impacts several aspects of telomere structure and function. Although Ku avidly binds DNA ends via a preformed channel, its ability to associate with telomeres via this mechanism could be challenged by factors known to bind directly to the chromosome terminus. This has led to uncertainty as to whether Ku itself binds directly to telomeric ends and whether end association is crucial for Ku's telomeric functions. To address these questions, we constructed DNA end binding–defective Ku heterodimers by altering amino acid residues in Ku70 and Ku80 that were predicted to contact DNA. These mutants continued to associate with their known telomere-related partners, such as Sir4, a factor required for telomeric silencing, and TLC1, the RNA component of telomerase. Despite these interactions, we found that the Ku mutants had markedly reduced association with telomeric chromatin and null-like deficiencies for telomere end protection, length regulation, and silencing functions. In contrast to Ku null strains, the DNA end binding defective Ku mutants resulted in increased, rather than markedly decreased, imprecise end-joining proficiency at an induced double-strand break. This result further supports that it was the specific loss of Ku's telomere end binding that resulted in telomeric defects rather than global loss of Ku's functions. The extensive telomere defects observed in these mutants lead us to propose that Ku is an integral component of the terminal telomeric cap, where it promotes a specific architecture that is central to telomere function and maintenance. The telomeric cap modulates telomere replication and prevents natural chromosome ends from being processed as DNA double-strand breaks (DSBs). In multiple species, including budding yeast, a detailed picture exists of the factors that comprise the telomeric cap and how they associate with telomeric DNA. It is less clear where to place Ku, a conserved heterodimer involved in multiple aspects of telomere biology and DSB repair. Although Ku avidly binds DNA ends, its access to telomeric ends might be restricted by telomere binding proteins and/or higher-order telomere structure. Ku might also be recruited to telomeres via its telomere-associated binding partners. Here, we address whether Ku loads directly onto telomeric ends and whether direct DNA binding is crucial for its telomeric functions. Using structure-guided mutagenesis, we generated end binding–defective yeast Ku heterodimers that retained the ability to associate with Ku's known telomeric binding partners. These end binding–defective heterodimers showed a dramatic reduction in telomere association and were defective for all of Ku's telomeric functions. Our findings indicate that Ku is indeed a component of the telomere cap and that its loading onto telomeric ends is crucial for its telomeric functions and, perhaps, a specific telomere architecture.
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DOI：
10.1126/science.1170633
发表时间：
2009-11-13
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
de Lange T
通讯作者：
de Lange T
影响因子：
4.5
作者：
Bonetti D;Clerici M;Anbalagan S;Martina M;Lucchini G;Longhese MP
通讯作者：
Longhese MP
影响因子：
21.3
作者：
Liu, D;Safari, A;Zhou, SY
通讯作者：
Zhou, SY
影响因子：
16.8
作者：
Fisher, TS;Taggart, AKP;Zakian, VA
通讯作者：
Zakian, VA
影响因子：
10.5
作者：
Hsu, HL;Gilley, D;Chen, DJ
通讯作者：
Chen, DJ