Institut Jacques Monod, CNRS UMR 7592, University of Paris - Diderot, 15 rue Hélène Brion, 75205 Paris Cedex 13, France.
Nucleic Acids Res. 2011 May;39(10):4275-83. doi: 10.1093/nar/gkr008. Epub 2011 Jan 25.
During cruciform extrusion, a DNA inverted repeat unwinds and forms a four-way junction in which two of the branches consist of hairpin structures obtained by self-pairing of the inverted repeats. Here, we use single-molecule DNA nanomanipulation to monitor in real-time cruciform extrusion and rewinding. This allows us to determine the size of the cruciform to nearly base pair accuracy and its kinetics with second-scale time resolution. We present data obtained with two different inverted repeats, one perfect and one imperfect, and extend single-molecule force spectroscopy to measure the torque dependence of cruciform extrusion and rewinding kinetics. Using mutational analysis and a simple two-state model, we find that in the transition state intermediate only the B-DNA located between the inverted repeats (and corresponding to the unpaired apical loop) is unwound, implying that initial stabilization of the four-way (or Holliday) junction is rate-limiting. We thus find that cruciform extrusion is kinetically regulated by features of the hairpin loop, while rewinding is kinetically regulated by features of the stem. These results provide mechanistic insight into cruciform extrusion and help understand the structural features that determine the relative stability of the cruciform and B-form states.
在十字形挤出过程中，DNA 反向重复序列解旋并形成四链结，其中两个分支由反向重复序列自身配对形成的发夹结构组成。在这里，我们使用单分子 DNA 纳米操作来实时监测十字形挤出和重绕。这使我们能够以近乎碱基对精度确定十字形的大小及其动力学，具有二级时间分辨率。我们提供了使用两个不同的反向重复序列获得的数据，一个是完美的，一个是不完美的，并扩展了单分子力谱来测量十字形挤出和重绕动力学的扭矩依赖性。通过突变分析和简单的两态模型，我们发现，在过渡状态中间，只有位于反向重复序列之间的 B-DNA（对应于未配对的顶端环）被解旋，这意味着四链结（或霍利迪）的初始稳定是限速的。因此，我们发现十字形挤出动力学受到发夹环特征的调节，而重绕动力学受到茎特征的调节。这些结果为十字形挤出提供了机制上的见解，并有助于理解决定十字形和 B 型状态相对稳定性的结构特征。
