Characterization of a K+-Induced Conformational Switch in a Human Telomeric DNA Oligonucleotide Using 2-Aminopurine Fluorescence
Characterization of a K+-Induced Conformational Switch in a Human Telomeric DNA Oligonucleotide Using 2-Aminopurine Fluorescence
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DOI：
10.1021/bi901357r
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发表时间：
2010-01-12
期刊：
影响因子：
2.9
通讯作者：
Chaires, Jonathan B.
中科院分区：
文献类型：
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作者：
Gray, Robert D.;Petraccone, Luigi;Chaires, Jonathan B.
人类端粒DNA由DNA序列d(GGGTTA)的串联重复序列组成。寡核苷酸端粒模型如d[A(GGGTTA)(3)GGG](Tel22)以阳离子依赖的方式折叠成由d(TTA)环连接的堆积G-季度组成的四链结构。核磁共振研究表明，在Na+溶液中，Tel22形成了四条反平行链的“篮子”拓扑；相比之下，在K+溶液中，Tel22由未知拓扑的混合物组成。我们以前对Tel22和类似的端粒类似物折叠机制的研究利用了270到325 nm之间的紫外吸收变化，主要报道了G-四联体的形成和堆积，结果表明，与适当的阳离子混合后，四链的形成在毫秒内发生。在本研究中，我们使用Tel22衍生物来评估特定环的折叠动力学和平衡，其中da残基被荧光报告碱基2-氨基嘌呤(2-AP)串联取代。通过2-AP荧光变化来评估Na+或K+诱导的Tel22折叠至少由三个动力学步骤组成，时间常数从毫秒到几百秒不等。Tel22折叠的依赖于Na+的平衡滴定可以近似为一个合作的两态过程。相反，依赖于K+的折叠曲线是双相的，表明1 mM和30 mM K+中存在不同的构象系综。这一结论得到了核磁共振氢谱的证实。分子动力学模拟揭示了位于DA1附近的Tel22上的K+结合口袋，该口袋专用于所谓的杂交-1构象，其中链1平行排列。这种可能的拓扑特异性结合位点的存在表明V可能通过调节四链三级结构而在调节端粒的构象和功能中发挥变构作用。
Human telomeric DNA consists of tandem repeats of the DNA sequence d(GGGTTA). Oligodeoxynucleotide telomere models such as d[A(GGGTTA)(3)GGG] (Tel22) fold in a cation-dependent manner into quadruplex Structures consisting of stacked G-quarters linked by d(TTA) loops. NMR has shown that in Na+ solutions Tel22 forms it "basket" topology of four antiparallel strands; in contrast, Tel22 in K+ solutions consists of a Mixture of unknown topologies. Our previous studies on the mechanism of folding of Tel22 and similar telomere analogues utilized changes ill UV absorption between 270 and 325 nm that report primarily on G-quartet formation and stacking showed that quadruplex formation Occurs Within milliseconds upon mixing with an appropriate cation. In this Study, we assess the dynamics and equilibria of folding of specific loops by using Tel22 derivatives in which the dA residues were serially substituted with the fluorescent reporter base, 2-aminopurine (2-AP). Tel22 folding Induced by Na+ or K+ assessed by changes in 2-AP fluorescence consists of at least three kinetic steps with time constants spanning a range from milliseconds to several hundred seconds. Na+-dependent equilibrium titrations of Tel22 folding could be approximated as a cooperative two-state process. In contrast, K+-dependent folding Curves were biphasic, revealing that different conformational ensembles are present in 1 and 30 mM K+. This conclusion wits confirmed by H-1 NMR. Molecular dynamics simulations revealed a K+ binding pocket in Tel22 located near dA1 that is specific for the so-called hybrid-1 conformation in which strand 1 is in a parallel arrangement. The possible presence of this topologically specific binding site suggests that V may play an allosteric role in regulating telomere conformation and function by modulating quadruplex tertiary structure.