Hüsler P L, Klump H H
Department of Biochemistry, University of Cape Town, Rondebosch, Republic of South Africa.
Arch Biochem Biophys. 1995 Sep 10;322(1):149-66. doi: 10.1006/abbi.1995.1447.
We have designed a Hoogsteen (HG) triple-helical three-way junction (ternary complex) constructed from three 33-mer oligonucleotides based on the same subset of sequences used for the Watson-Crick (WC) triple-helical three-way junction, characterized previously (P. L. Hüsler and H. H. Klump (1994) Arch. Biochem. Biophys., 313, 29-38). The junction differs primarily in the assembly of the branch point and the ends of the arms. The three oligonucleotides can each fold into a WC hairpin, linked by a four-member cytosine loop, each containing a homo-pyrimidine 10-mer single-strand extension. On lowering the pH (between 6 and 4), the extensions mutually associate to one of the other hairpins via Hoogsteen (HG) hydrogen bonding. Collectively, this process results in the formation of the branch point and the triple-helical arms. The HG triple-helical three-way junction is characterized by gel electrophoresis, circular dichroism, uv melting, and differential scanning calorimetry. The junction undergoes thermal unfolding in two distinct temperature regions. In the temperature range 15 to 50 degrees C loss of HG base pairing results in the dissociation of the three-way junction. Between 55 and 95 degrees C the resulting hairpins undergo further successive unfolding. The overall calorimetric unfolding enthalpy and entropy changes associated with the loss of HG base pairing are approximately equal to the sum of the enthalpy and entropy changes associated with the dissociation of the HG base pairing in the isolated arms (170.6 kcal.mol-1; 540.1 cal.mol-1.K-1). It is apparent from these results that in the proximity of the branch point the structure is not perturb or strain. This result is contrary to the results obtained for the WC triple-helical three-way and for three-way junctions constructed from canonical double-helical DNA. Complete folding of the junction requires either high Na+ (600 mM) ion concentrations or 40-60 mM Mg2+.
我们设计了一种霍格斯坦（HG）三螺旋三向连接体（三元复合物），它由三条33聚体寡核苷酸构建而成，这些寡核苷酸基于先前已表征的用于沃森-克里克（WC）三螺旋三向连接体的相同序列子集（P. L. 许斯勒和H. H. 克伦普（1994年）《生物化学与生物物理学档案》，313卷，29 - 38页）。该连接体的主要差异在于分支点和臂末端的组装方式。这三条寡核苷酸各自都能折叠成一个WC发夹结构，通过一个四聚体胞嘧啶环相连，每个发夹结构都包含一个同型嘧啶10聚体单链延伸部分。当降低pH值（在6到4之间）时，这些延伸部分通过霍格斯坦（HG）氢键相互与另一个发夹结构缔合。总体而言，这个过程导致了分支点和三螺旋臂的形成。HG三螺旋三向连接体通过凝胶电泳、圆二色性、紫外熔解和差示扫描量热法进行表征。该连接体在两个不同的温度区域发生热解折叠。在15至50摄氏度的温度范围内，HG碱基对的丧失导致三向连接体解离。在55至95摄氏度之间，形成的发夹结构会进一步连续解折叠。与HG碱基对丧失相关的总热解折叠焓变和熵变大约等于与孤立臂中HG碱基对解离相关的焓变和熵变之和（170.6千卡·摩尔⁻¹；540.1卡·摩尔⁻¹·开⁻¹）。从这些结果可以明显看出，在分支点附近，结构没有受到扰动或应变。这个结果与从经典双螺旋DNA构建的WC三螺旋三向连接体和三向连接体所得到的结果相反。连接体的完全折叠需要高浓度的Na⁺（600 mM）离子或40 - 60 mM Mg²⁺。
