Probing the mechanism of recognition of ssDNA by the Cdc13-DBD.
Probing the mechanism of recognition of ssDNA by the Cdc13-DBD.
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DOI：
10.1093/nar/gkn017
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发表时间：
2008-03
影响因子：
14.9
通讯作者：
Wuttke DS
中科院分区：
文献类型：
--
作者：
Eldridge AM;Wuttke DS
酿酒酵母Cdc 13蛋白与端粒上保守的富含G的单链突出端紧密特异性结合，在端粒末端保护和长度调节中发挥重要作用。Cdc 13的200个残基的DNA结合结构域（Cdc 13-DBD）以3 pM的亲和力和对5′端三个碱基（下划线）的特异性结合酵母端粒序列[Tel 11，d（GTGTGGGTGTG）]的11聚体单链代表。的Cdc 13-DBD绑定到Tel 11的结构揭示了一个大的，主要是芳香族蛋白质接口与几个不寻常的功能。DNA采用不规则的延伸结构，并且结合界面包括OB-折叠的链β2-β3（L2-3）之间的长（约30个氨基酸）结构环。为了研究ssDNA结合的机制，我们使用NMR光谱研究了Cdc 13-DBD的自由态和结合态。化学位移的变化表明，基本拓扑结构的域，包括L2-3，基本上是完整的在自由状态。然而，在缓慢和中间的时间尺度动态的变化，发生在L2-3，而远离DNA界面的构象变化表明在结合亲和力和特异性的“热点”结合诱导适合机制。这些数据点的整体结合机制很好地适应酵母端粒的异质性。
The Saccharomyces cerevisiae protein Cdc13 tightly and specifically binds the conserved G-rich single-stranded overhang at telomeres and plays an essential role in telomere end-protection and length regulation. The 200 residue DNA-binding domain of Cdc13 (Cdc13-DBD) binds an 11mer single-stranded representative of the yeast telomeric sequence [Tel11, d(GTGTGGGTGTG)] with a 3 pM affinity and specificity for three bases (underlined) at the 5′ end. The structure of the Cdc13-DBD bound to Tel11 revealed a large, predominantly aromatic protein interface with several unusual features. The DNA adopts an irregular, extended structure, and the binding interface includes a long (∼30 amino acids) structured loop between strands β2-β3 (L2–3) of an OB-fold. To investigate the mechanism of ssDNA binding, we studied the free and bound states of Cdc13-DBD using NMR spectroscopy. Chemical shift changes indicate that the basic topology of the domain, including L2–3, is essentially intact in the free state. Changes in slow and intermediate time scale dynamics, however, occur in L2–3, while conformational changes distant from the DNA interface suggest an induced fit mechanism for binding in the ‘hot spot’ for binding affinity and specificity. These data point to an overall binding mechanism well adapted to the heterogeneous nature of yeast telomeres.
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