Déclais Anne-Cécile, Liu Jia, Freeman Alasdair D J, Lilley David M J
Cancer Research UK Nucleic Acid Structure Research Group, MSI/WTB Complex, The University of Dundee.
J Mol Biol. 2006 Jun 23;359(5):1261-76. doi: 10.1016/j.jmb.2006.04.037. Epub 2006 May 2.
Resolving enzymes bind highly selectively to four-way DNA junctions, but the mechanism of this structural specificity is poorly understood. In this study, we have explored the role of interactions between the dimeric enzyme and the helical arms of the junction, using junctions with either shortened arms, or circular permutation of arms. We find that DNA-protein contacts in the arms containing the 5' ends of the continuous strands are very important, conferring a significant level of sequence discrimination upon both the choice of conformer and the order of strand cleavage. We have exploited these properties to obtain hydroxyl radical footprinting data on endonuclease I-junction complexes that are not complicated by the presence of alternative conformers, with results that are in good agreement with the arm permutation and shortening experiments. Substitution of phosphate groups at the center of the junction reveals the importance of electrostatic interactions at the point of strand exchange in the complex. Our data show that the form of the complex between endonuclease I and a DNA junction depends on the core of the junction and on interactions with the first six base-pairs of the arms containing the 5' ends of the continuous strands.
切割酶与四链DNA连接点具有高度选择性结合，但这种结构特异性的机制却鲜为人知。在本研究中，我们利用臂缩短或臂环形排列的连接点，探究了二聚体酶与连接点螺旋臂之间相互作用的作用。我们发现，包含连续链5'端的臂中的DNA-蛋白质接触非常重要，这在构象选择和链切割顺序上都赋予了显著水平的序列辨别能力。我们利用这些特性获得了关于核酸内切酶I-连接点复合物的羟基自由基足迹数据，这些数据不会因存在替代构象而变得复杂，其结果与臂排列和缩短实验结果高度吻合。连接点中心磷酸基团的取代揭示了复合物中链交换点处静电相互作用的重要性。我们的数据表明，核酸内切酶I与DNA连接点之间复合物的形式取决于连接点的核心以及与包含连续链5'端的臂的前六个碱基对的相互作用。
