Makube N, Klump H, Pikkemaat J, Altona C
Department of Biochemistry, University of Cape Town, Private Bag Rondebosch, 7700, Republic of South Africa.
Arch Biochem Biophys. 1999 Apr 1;364(1):53-60. doi: 10.1006/abbi.1998.1061.
We have investigated the thermodynamic properties of two homologous DNA four-way junctions, J4 and J4M, based on 46-mer linear DNA molecules. J4 and J4M have the same base sequence with the only difference that the latter contains an uncharged methylene-acetal linkage, -O3'-CH2-O5', instead of the phosphodiester linkage, -O3'-PO2-O5'-, between the residues T18 and C19. The comparison of the thermal unfolding of the J4 junction and J4M junction serves to investigate the effect of the uncharged methylene-acetal linkage on the stability of the junction. Our analysis is based on CD, UV absorbance spectroscopy, DSC, and chemical footprinting. The aim is to characterize in detail the structure and stability of the junctions. As demonstrated before by NMR, in the presence of 5 mM MgCl2 +/- 50 mM NaCl, both J4 and J4M form a complete four-way junction. This is now evidenced by protection from OsO4 cleavage (chemical footprinting). We can assume that full base pairing occurs throughout the arms even at the center of the junction. CD spectra suggest that the helices within the junctions adopt the regular B-DNA conformation. Almost identical melting temperatures and unfolding enthalpies are obtained for J4 and J4M both by UV and DSC. Furthermore, the Van't Hoff enthalpy (DeltaHVH) derived from UV melting equals the calorimetric enthalpy (DeltaHcal), which means that the melting process of the structures proceeds in a two-state manner. All results taken together support the conclusion that there are no major conformational and energetic differences between J4 and J4M. The inclusion of the uncharged methylene-acetal group into the junction has no effect on its stability.
我们基于46聚体线性DNA分子研究了两个同源DNA四链体连接体J4和J4M的热力学性质。J4和J4M具有相同的碱基序列，唯一的区别在于，在残基T18和C19之间，后者含有一个不带电荷的亚甲基缩醛连接键-O3'-CH2-O5'，而不是磷酸二酯连接键-O3'-PO2-O5'-。对J4连接体和J4M连接体的热解链进行比较，有助于研究不带电荷的亚甲基缩醛连接键对连接体稳定性的影响。我们的分析基于圆二色光谱（CD）、紫外吸收光谱、差示扫描量热法（DSC）和化学足迹法。目的是详细表征连接体的结构和稳定性。如之前核磁共振（NMR）所证明的，在存在5 mM MgCl2 ± 50 mM NaCl的情况下，J4和J4M均形成完整的四链体连接体。现在通过对锇酸（OsO4）切割的保护（化学足迹法）证明了这一点。我们可以假定，即使在连接体的中心，整个臂中也会发生完全的碱基配对。CD光谱表明，连接体内的螺旋采用规则的B-DNA构象。通过紫外光谱和DSC获得的J4和J4M的解链温度和解链焓几乎相同。此外，由紫外解链得到的范特霍夫焓（ΔHVH）等于量热焓（ΔHcal），这意味着结构的解链过程以两态方式进行。综合所有结果支持以下结论：J4和J4M之间在构象和能量上没有重大差异。在连接体中引入不带电荷的亚甲基缩醛基团对其稳定性没有影响。
