Mariappan S V, Silks L A, Chen X, Springer P A, Wu R, Moyzis R K, Bradbury E M, Garcia A E, Gupta G
Theoretical Biology and Biophysics, Los Alamos National Laboratory, NM 87545, USA.
J Biomol Struct Dyn. 1998 Feb;15(4):723-44. doi: 10.1080/07391102.1998.10508988.
Highly polymorphic DNA triplet repeats, (CAG)n, are located inside the first exon of the Huntington's disease gene. Inordinate expansion of this repeat is correlated with the onset and progression of the disease. NMR spectroscopy, gel electrophoresis, digestion by single-strand specific P1 enzyme, and in vitro replication assay have been used to investigate the structural basis of (CAG)n expansion. Nondenaturing gel electrophoresis and 1D 1H NMR studies of (CAG)5 and (CAG)6 reveal the presence of hairpins and mismatched duplexes as the major and minor populations respectively. However, at high DNA concentrations (i.e., 1.0-2.0 mM that is typically required for 2D NMR experiments) both (CAG)5 and (CAG)6 exist predominantly in mismatched duplex forms. Mismatched duplex structures of (CAG)5 and (CAG)6 are useful, because they adequately model the stem of the biologically relevant hairpins formed by (CAG)n. We, therefore, performed detailed NMR spectroscopic studies on the duplexes of (CAG)5 and (CAG)6. We also studied a model duplex, (CGCAGCG)2 that contains the underlined building block of the duplex. This duplex shows the following structural characteristics: (i) all the nucleotides are in (C2'-endo, anti) conformations, (ii) mismatched A x A base pairs are flanked by two Watson-Crick G x C base pairs and (iii) A x A base pairs are stably stacked (and intra-helical) and are formed by a single N6-H--N1 hydrogen bond. The nature of A x A pairing is confirmed by temperature-dependent HMQC and HMQC-NOESY experiments on the [(CA*G)5]2 duplex where the adenines are 15N-labeled at N6. Temperature- and pH-dependent imino proton spectra, nondenaturing electrophoresis, and P1 digestion data demonstrate that under a wide range of solution conditions longer (CAG)n repeats (n> or =10) exist exclusively in hairpin conformation with two single-stranded loops. Finally, an in vitro replication assay with (CAG)8,21 inserts in the M13 single-stranded DNA templates shows a replication bypass for the (CAG)21 insert but not for the (CAG)8 insert in the template. This demonstrates that for a sufficiently long insert (n=21 in this case), a hairpin is formed by the (CAG)n even in presence of its complementary strand. This observation implies that the formation of hairpin by the (CAG)n may cause slippage during replication and thus may explain the observed length polymorphism.
高度多态的DNA三联体重复序列（CAG）n位于亨廷顿舞蹈病基因的第一个外显子内。该重复序列的过度扩增与疾病的发生和发展相关。核磁共振光谱、凝胶电泳、单链特异性P1酶消化以及体外复制分析已被用于研究（CAG）n扩增的结构基础。对（CAG）5和（CAG）6进行的非变性凝胶电泳和一维氢核磁共振研究分别揭示了发夹结构和错配双链体作为主要和次要群体的存在。然而，在高DNA浓度下（即二维核磁共振实验通常所需的1.0 - 2.0 mM），（CAG）5和（CAG）6都主要以错配双链体形式存在。（CAG）5和（CAG）6的错配双链体结构是有用的，因为它们充分模拟了由（CAG）n形成的生物学相关发夹结构的茎部。因此，我们对（CAG）5和（CAG）6的双链体进行了详细的核磁共振光谱研究。我们还研究了一个模型双链体（CGCAGCG）2，它包含了双链体中带下划线的结构单元。该双链体具有以下结构特征：（i）所有核苷酸均处于（C2'-内，反式）构象，（ii）错配的A×A碱基对两侧是两个沃森-克里克G×C碱基对，（iii）A×A碱基对稳定堆积（且在螺旋内部），并由单个N6-H--N1氢键形成。通过对腺嘌呤在N6处用15N标记的[(CA*G)5]2双链体进行温度依赖性HMQC和HMQC-NOESY实验，证实了A×A配对的性质。温度和pH依赖性亚氨基质子光谱、非变性电泳以及P1消化数据表明，在广泛的溶液条件下，较长的（CAG）n重复序列（n≥10）仅以具有两个单链环的发夹构象存在。最后，在M13单链DNA模板中插入（CAG）8,21进行的体外复制分析表明，模板中的（CAG）21插入片段存在复制旁路，而（CAG）8插入片段则没有。这表明对于足够长的插入片段（在这种情况下n = 21），即使存在互补链，（CAG）n也会形成发夹结构。这一观察结果意味着（CAG）n形成发夹结构可能会在复制过程中导致滑动，从而可能解释所观察到的长度多态性。
