Slow motion in the CAA•TTG sequence of a DNA decamer duplex studied by NMR
Slow motion in the CAA•TTG sequence of a DNA decamer duplex studied by NMR
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DOI：
10.1021/bi0103260
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发表时间：
2001-06-19
期刊：
影响因子：
2.9
通讯作者：
James, TL
中科院分区：
文献类型：
--
作者：
Kojima, C;Ulyanov, NB;James, TL
在DNA双链体中，嘧啶-嘌呤步骤被认为是柔性的或构象不稳定的。事实上，几种DNA晶体结构表现出多种CpA构象。TpG步骤。问题是，这种结构灵活性是否伴随着动力灵活性，即，这是一个关于构象间能垒的问题。除了TpA步骤，在微秒到毫秒的时间尺度上的慢动作还没有被检测到的双重直到现在。在本研究中，这样的慢运动进行了研究，H-1，C-13，和N-15 NMR弛豫测量的DNA十聚体d（CATTTGCATC）。d（GATGCAAATG）。对于每个腺苷和鸟苷残基，DNA十聚体富集有15%C-13和98%N-15同位素。有三条证据支持CAA中慢动作的概念。TTG部分。N ~（15）弛豫分析表明，鸟苷亚胺NH基的序参量S ~（-2）约为0.8，与该寡聚体CH基的序参量S ~（-2）相似。TTG序列显示存在大幅度运动。CAA中腺苷H8质子的信号。在二维H-1 NOESY谱中，TTG序列被加宽，这也表明慢运动的存在。除了小于其他腺嘌呤残基之外，H-1 T-2值对于ATTTG中的所有腺苷H8信号表现出磁场强度依赖性。CAAAT区域，表明CAA中第一个腺苷处的缓慢运动更加明显。TTG序列，但在CAAAT上延伸。ATTTG区域。通过H-1、C-13和N-15 T-1 ρ值的脉冲场强依赖性进一步检验了这一现象。H-1和C-13 T-1 rho值显示出脉冲场强依赖性，但N-15 T-1 rho没有。假设一个两个网站的交换过程中，交换时间常数为20-300 μ s的CAA序列中的第一个腺苷估计。这一运动的确切性质仍不清楚。
In DNA duplexes, pyrimidine-purine steps are believed to be flexible or conformationally unstable. Indeed, several DNA crystal structures exhibit a multitude of conformations for CpA . TpG steps. The question arises of whether this structural flexibility is accompanied by dynamical flexibility, i.e., a question pertaining to the energy barrier between conformations. Except for TpA steps, slow motions on the microsecond-to-millisecond time scale have not been detected in duplexes until now. In the present study, such slow motion was investigated by H-1, C-13, and N-15 NMR relaxation measurements on a DNA decamer d(CATTTGCATC).d(GATGCAAATG). The DNA decamer was enriched with 15% C-13 and 98% N-15 isotopes for each adenosine and guanosine residue. Three lines of evidence support the notion of slow motion in the CAA . TTG moiety. Analysis of N-15 relaxation showed that the order parameter, S-2, of guanosine imino NH groups was about 0.8, similar to that of CH groups for this oligomer, The strong temperature dependence of guanosine NH S-2 in the CAA . TTG sequence indicated the presence of a large-amplitude motion. Signals of adenosine H8 protons in the CAA . TTG sequence were broadened in 2D H-1 NOESY spectra, which also suggested the existence of slow motion. As well as being smaller than for other adenine residues, the H-1 T-2 values exhibited a magnetic field strength dependence for all adenosine H8 signals in the ATTTG . CAAAT region, suggesting slow motions more pronounced at the first adenosine in the CAA . TTG sequence but extending over the CAAAT . ATTTG region. This phenomenon was further examined by the pulse field strength dependence of the H-1, C-13, and N-15 T-1 rho values. H-1 and C-13 T-1 rho values showed a pulse field strength dependence, but N-15 T-1 rho did not. Assuming a two-site exchange process, an exchange time constant of 20-300 mus was estimated for the first adenosine in the CAA sequence. The exact nature of this motion remains unknown.