Solution formation of Holliday junctions in inverted-repeat DNA sequences.
Solution formation of Holliday junctions in inverted-repeat DNA sequences.
复制标题
反向重复 DNA 序列中霍利迪连接的溶液形成。
DOI：
10.1021/bi052129x
复制
发表时间：
2006
期刊：
影响因子：
2.9
通讯作者：
Demeler,Borries
中科院分区：
文献类型：
--
作者：
Hays,FranklinA;Schirf,Virgil;Ho,PShing;Demeler,Borries
现在，通过对称（反向重复）DNA 序列中的单晶 X 射线衍射，霍利迪连接体的结构已在原子水平上得到了很好的表征。然而，问题在于这些四链复合物在溶液中的形成是否真正以所提出的方式依赖于序列，或者是结晶过程的人为产物，因此与同源重组和重组依赖性细胞过程中这一中心中间体的行为无关。在这里，我们应用分析超速离心来证明序列 d(CCGGTACCGG)（以堆叠 X 形式的连接结晶）在溶液中组装成四链连接，其方式取决于 DNA 和阳离子浓度，并在 100−200 μM DNA 双链体时在连接和双链体形式之间建立平衡。相比之下，已结晶为B-DNA的序列d(CCGCTAGCGG)在所测试的所有DNA和盐浓度下仅采用双螺旋形式。因此，ACC三核苷酸核心现在被证明对于晶体和溶液中霍利迪连接的形成很重要，并且可以估计贡献大约-4 kcal/mol以稳定反向重复序列中的这种重组中间体。
The structure of Holliday junctions has now been well characterized at the atomic level through single-crystal X-ray diffraction in symmetric (inverted-repeat) DNA sequences. At issue, however, is whether the formation of these four-stranded complexes in solution is truly sequence dependent in the manner proposed or is an artifact of the crystallization process and, therefore, has no relevance to the behavior of this central intermediate in homologous recombination and recombination-dependent cellular processes. Here, we apply analytical ultracentrifugation to demonstrate that the sequence d(CCGGTACCGG), which crystallizes in the stacked-X form of the junction, assembles into four-stranded junctions in solution in a manner that is dependent on the DNA and cation concentrations, with an equilibrium established between the junction and duplex forms at 100−200 μM DNA duplex. In contrast, the sequence d(CCGCTAGCGG), which has been crystallized as B-DNA, is seen to adopt only the double-helical form at all DNA and salt concentrations that were tested. Thus, the ACC trinucleotide core is now shown to be important for the formation of Holliday junctions in both crystals and in solution and can be estimated to contribute approximately −4 kcal/mol to stabilizing this recombination intermediate in inverted-repeat sequences.