Coordinated Interactions of Multiple POT1-TPP1 Proteins with Telomere DNA
Coordinated Interactions of Multiple POT1-TPP1 Proteins with Telomere DNA
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DOI：
10.1074/jbc.m113.471896
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发表时间：
2013-06-07
影响因子：
4.8
通讯作者：
Taylor, Derek J.
中科院分区：
文献类型：
--
作者：
Corriveau, Mark;Mullins, Michael R.;Taylor, Derek J.
端粒是保护真核生物染色体末端免受降解、端到端融合事件和参与DNA损伤反应的大分子核蛋白复合物。然而，人们对这种重要的dna -蛋白质复合物的组装知之甚少。在脊椎动物中，端粒DNA由重复的双链序列5‘-TTAGGG-3’组成，随后是具有相同序列的单链DNA悬垂。双链和单链区域都被端粒末端结合蛋白（包括POT1和TPP1）高特异性包裹，这些蛋白作为异源二聚体结合到单链端粒DNA上。多个POT1-TPP1蛋白必须完全包裹单链端粒DNA才能形成功能性端粒。为了更好地理解多重结合的机制，我们突变或删除了位于单链端粒DNA中相邻POT1-TPP1识别位点之间的两个鸟苷核苷酸，这些核苷酸对于多重POT1-TPP1结合事件并不需要。圆二色性表明，来自天然端粒序列的光谱具有g -四重体二级结构的特征，而改变的端粒序列则没有这些特征。然而，与野生型端粒序列相比，改变的端粒链促进了多个POT1-TPP1蛋白的协同装载。最后，我们发现一个含有端粒序列的48个核苷酸的DNA在包被POT1-TPP1蛋白时比未包被时更容易被核酸酶酶切。总之，这些数据表明，POT1-TPP1以一种协调的方式结合端粒DNA，以促进核蛋白复合物的组装，使其进入一种更易于酶活性的状态。
Telomeres are macromolecular nucleoprotein complexes that protect the ends of eukaryotic chromosomes from degradation, end-to-end fusion events, and from engaging the DNA damage response. However, the assembly of this essential DNA-protein complex is poorly understood. Telomere DNA consists of the repeated double-stranded sequence 5'-TTAGGG-3' in vertebrates, followed by a single-stranded DNA overhang with the same sequence. Both double-and single-stranded regions are coated with high specificity by telomere end-binding proteins, including POT1 and TPP1, that bind as a heterodimer to single-stranded telomeric DNA. Multiple POT1-TPP1 proteins must fully coat the single-stranded telomere DNA to form a functional telomere. To better understand the mechanism of multiple binding, we mutated or deleted the two guanosine nucleotides residing between adjacent POT1-TPP1 recognition sites in single-stranded telomere DNA that are not required for multiple POT1-TPP1 binding events. Circular dichroism demonstrated that spectra from the native telomere sequence are characteristic of a G-quadruplex secondary structure, whereas the altered telomere sequences were devoid of these signatures. The altered telomere strands, however, facilitated more cooperative loading of multiple POT1-TPP1 proteins compared with the wild-type telomere sequence. Finally, we show that a 48-nucleotide DNA with a telomere sequence is more susceptible to nuclease digestion when coated with POT1-TPP1 proteins than when it is left uncoated. Together, these data suggest that POT1-TPP1 binds telomeric DNA in a coordinated manner to facilitate assembly of the nucleoprotein complexes into a state that is more accessible to enzymatic activity.