Duckett D R, Lilley D M
Department of Biochemistry, The University, Dundee, U.K.
J Mol Biol. 1991 Sep 5;221(1):147-61. doi: 10.1016/0022-2836(91)80211-c.
Heteroduplex formation between imperfectly homologous DNA sequences may result in the formation of a four-way junction at which non-Watson-Crick base mismatches are present at the point of strand exchange. This raises the question of the effect of such mismatches on the structure and stability of these potential recombination intermediates. We have constructed a series of four-way DNA junctions containing single-base mismatches, and have studied the structure of the junctions by means of gel electrophoresis and chemical modification. We observed a range of effects on the structure of the junction, ranging from almost total abolition of folding through to normal accommodation into the folded structure. In some cases we observed gel electrophoretic data consistent with a dynamic equilibrium between folded and unfolded conformations, and in general the folded form was favoured at higher concentrations of cation. The effects of single mismatches on the structure of the four-way junction may be summarized in terms of: (1) the nature of the mismatch, where we note a correlation between the thermal stability of a given mismatch and its ability to be accommodated into a folded junction; or (2) the sequence context, where the effect of a given mismatch on the structure of a junction depends on the neighbouring base-pairs. These factors are illustrated by a junction, containing a C.A mismatch, that adopted alternate isomeric conformations dependent upon pH; as the state of protonation of the mispair changed, the structure was altered along with the interaction with neighbouring base-pairs. Most base mismatches may be accommodated into the folded stacked X-conformation of the four-way junction, but many require elevated cation concentration to permit the folding process to proceed. Some mismatches were found to be extremely destabilizing.
不完全同源的DNA序列之间形成异源双链体可能会导致形成一个四向连接点，在链交换点处存在非沃森-克里克碱基错配。这就引发了这样一个问题，即这种错配对这些潜在重组中间体的结构和稳定性有何影响。我们构建了一系列包含单碱基错配的四向DNA连接点，并通过凝胶电泳和化学修饰研究了连接点的结构。我们观察到连接点结构受到一系列影响，范围从折叠几乎完全消失到正常纳入折叠结构。在某些情况下，我们观察到凝胶电泳数据与折叠和未折叠构象之间的动态平衡一致，并且一般来说，在较高阳离子浓度下折叠形式更受青睐。单碱基错配对四向连接点结构的影响可以概括为：（1）错配的性质，我们注意到给定错配的热稳定性与其纳入折叠连接点的能力之间存在相关性；或者（2）序列背景，给定错配对接合点结构的影响取决于相邻碱基对。一个包含C.A错配的连接点说明了这些因素，该连接点根据pH采用交替的异构构象；随着错配碱基对的质子化状态改变，结构以及与相邻碱基对的相互作用也会改变。大多数碱基错配可以纳入四向连接点的折叠堆叠X构象，但许多需要提高阳离子浓度才能使折叠过程进行。发现一些错配极具不稳定作用。
