Pöhler J R, Duckett D R, Lilley D M
Department of Biochemistry, The University, Dundee, U.K.
J Mol Biol. 1994 Apr 22;238(1):62-74. doi: 10.1006/jmbi.1994.1268.
We have investigated the structure of the four-way helical DNA junction containing a single covalent discontinuity (nick) in one strand. These could result from either unitary strand exchange processes, or the action of nucleases upon a complete junction. We have employed gel electrophoresis methods to study the global configuration of arms in these junctions. We find that the junction carrying a nick in one strand undergoes a folding process in the presence of magnesium ion concentrations greater than 200 microM. Comparison of the electrophoretic mobilities of the six possible derivative junctions with two long and two shortened arms suggests that the folding occurs by coaxial stacking of pairs of helical arms, which is supported by the suppression of reactivity to osmium tetroxide of thymine bases at the centre of the junction. However, unlike the complete junction (i.e. the junction without nicked strands), the two stacked pairs of helices lie at a mutual angle of approximately 90 degrees. The folding process generates two kinds of strands; two continuous strands and two exchanging strands. Two isomers of the right-angled stacked structure are possible, depending on the selection of stacking partners; it appears that the critical factor determining the relative stabilities of these isomers is the location of the nick. Thus the nicked junctions fold into the isomer that locates the nick on the exchanging strand. However, if the nick is not located at the point of strand exchange, the junction reverts to the stacked X-structure of the complete junction, even if the nick is moved by a single base-pair. These results suggest that the exchanging strands may be significantly strained in the structure of the complete four-way junction, such that an interruption to the continuity at this position allows the two stacked helices to disengage, and rotate to an angle where the overall electrostatic repulsion may be lower.
我们研究了在一条链中含有单个共价间断（切口）的四链螺旋DNA连接体的结构。这些切口可能源于单一的链交换过程，或者核酸酶对完整连接体的作用。我们采用凝胶电泳方法来研究这些连接体中臂的整体构型。我们发现，在镁离子浓度大于200微摩尔时，一条链带有切口的连接体会经历折叠过程。对六种可能的衍生连接体（两条长臂和两条短臂）的电泳迁移率进行比较表明，折叠是通过螺旋臂对的同轴堆积发生的，这一点得到了连接体中心胸腺嘧啶碱基对四氧化锇反应性受抑制的支持。然而，与完整连接体（即没有带切口链的连接体）不同，两对堆叠的螺旋相互间呈约90度角。折叠过程产生两种链；两条连续链和两条交换链。根据堆积伙伴的选择，直角堆叠结构可能有两种异构体；似乎决定这些异构体相对稳定性的关键因素是切口的位置。因此，带切口的连接体折叠成将切口定位在交换链上的异构体。然而，如果切口不在链交换点处，连接体即使切口移动了一个碱基对，也会恢复到完整连接体的堆叠X结构。这些结果表明，在完整的四链连接体结构中，交换链可能会受到显著的张力，以至于在这个位置的连续性中断会使两个堆叠的螺旋分离，并旋转到一个整体静电排斥可能较低的角度。
