Identification of human TERT elements necessary for telomerase recruitment to telomeres.
Identification of human TERT elements necessary for telomerase recruitment to telomeres.
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DOI：
10.7554/elife.03563
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发表时间：
2014-10-01
期刊：
影响因子：
7.7
通讯作者：
Cech TR
中科院分区：
文献类型：
--
作者：
Schmidt JC;Dalby AB;Cech TR
人类染色体终止于端粒，端粒是由shelterin复合物结合的重复DNA序列。庇护蛋白保护染色体末端，防止DNA损伤机制的识别，并招募端粒酶。shelterin成分TPP 1的OB折叠结构域上的一片氨基酸（称为TEL片）对于将端粒招募到端粒至关重要。相比之下，端粒酶上与TPP 1 OB-折叠相互作用的位点尚未明确。在这项研究中，我们确定分离的功能突变的TEN结构域的人端粒酶逆转录酶（hTERT），破坏端粒酶与TPP 1在体内和体外的相互作用，但对端粒酶的催化活性的影响很小。TEN结构域突变与TEL-补丁中的补偿性电荷交换突变的抑制表明它们的关联是直接的。我们的研究结果定义了端粒酶募集到端粒所需的相互作用界面，这是开发这种相互作用的调节剂作为人类疾病治疗剂的重要一步。http://dx.doi.org/10.7554/eLife.03563.001在细胞核中，包含细胞遗传信息的DNA被包装成称为染色体的长结构。每当细胞分裂时，它的染色体就会复制。然而，负责复制DNA的蛋白质无法到达DNA链的末端，导致染色体逐渐缩短。为了确保这不会导致遗传信息丢失，每条染色体都以一段重复的DNA为末端，称为端粒。尽管每当DNA被复制时，端粒的末端就会丢失，但一种称为端粒酶的酶会取代丢失的序列，并抵消端粒的缩短。Shelterin是一种蛋白质复合物，它与端粒结合以保护端粒，并帮助端粒酶正确工作。Shelterin含有一个与端粒酶结合的特定位点，但这两种分子的人类版本如何相互结合尚不清楚。在端粒酶上发现了一个可能的相互作用位点，当突变时，它会阻止端粒酶正常工作。然而，由于该区域也参与染色体复制后端粒的延长，因此不能确定这些问题是由于端粒酶未能与shelterin结合而导致的。端粒酶是不寻常的;它既有RNA又有蛋白质成分。像所有其他蛋白质一样，端粒酶蛋白质是由一串氨基酸组成的。施密特等人发现，替换人端粒酶中的两个特定氨基酸可阻止其与shelterin结合。产生改良型端粒酶的细胞具有端粒缩短的染色体。然而，如果细胞也产生了被设计为与修饰的端粒酶结合的修饰的shelterin复合物，那么端粒长度是正常的。这表明端粒酶直接与shelterin相互作用，而不是通过“桥接”分子。编码shelterin和端粒酶的基因突变会导致许多人类疾病，癌症依赖于端粒酶的活性生长。因此，了解shelterin和端粒酶如何相互作用可能有助于设计可以恢复或破坏相互作用的药物，从而可用于治疗这些疾病。DOI：http://dx.doi.org/10.7554/eLife.03563.002网站
Human chromosomes terminate in telomeres, repetitive DNA sequences bound by the shelterin complex. Shelterin protects chromosome ends, prevents recognition by the DNA damage machinery, and recruits telomerase. A patch of amino acids, termed the TEL-patch, on the OB-fold domain of the shelterin component TPP1 is essential to recruit telomerase to telomeres. In contrast, the site on telomerase that interacts with the TPP1 OB-fold is not well defined. In this study, we identify separation-of-function mutations in the TEN-domain of human telomerase reverse transcriptase (hTERT) that disrupt the interaction of telomerase with TPP1 in vivo and in vitro but have very little effect on the catalytic activity of telomerase. Suppression of a TEN-domain mutation with a compensatory charge-swap mutation in the TEL-patch indicates that their association is direct. Our findings define the interaction interface required for telomerase recruitment to telomeres, an important step towards developing modulators of this interaction as therapeutics for human disease. DOI: http://dx.doi.org/10.7554/eLife.03563.001 In the nucleus of a cell, the DNA that contains the cell's genetic information is packaged into long structures called chromosomes. Every time a cell divides, its chromosomes are duplicated. However, the proteins that are responsible for copying the DNA cannot reach the very end of the DNA strand, causing the chromosomes to progressively shorten. To ensure that this does not cause genetic information to be lost, each chromosome ends in a repetitive stretch of DNA called a telomere. Though the end of the telomere is lost whenever the DNA is copied, an enzyme called telomerase replaces the sequence that has been lost and counteracts the shortening of the telomeres. Shelterin is a protein complex that binds to telomeres to protect them and also helps telomerase to work correctly. Shelterin contains a specific site that attaches to telomerase, but exactly how the human versions of these two molecules bind to each other is unknown. A possible interaction site had been identified on the telomerase, which, when mutated, stops the telomerase working properly. However, as this region is also involved in lengthening the telomeres after the chromosomes have duplicated, it is not certain that these problems result from the telomerase failing to bind to shelterin. The enzyme telomerase is unusual; it has both RNA and protein components. Like all other proteins, the telomerase protein is made up of strings of amino acids. Schmidt et al. discovered that replacing two specific amino acids in human telomerase prevents its binding to shelterin. Cells that produced the modified form of the telomerase had chromosomes with shortened telomeres. However, if the cells also produced modified versions of the shelterin complex that were designed to bind to the modified telomerase, telomere length was normal. This indicates that the telomerase interacts directly with shelterin, rather than through a ‘bridging’ molecule. Mutations in the genes coding for both shelterin and the telomerase enzyme cause a number of human diseases, and cancers rely on the activity of telomerases to grow. Knowing how shelterin and telomerase interact could therefore help to design drugs that may either restore or disrupt the interaction and therefore can be used to treat these diseases. DOI: http://dx.doi.org/10.7554/eLife.03563.002