Revealing the competition between peeled ssDNA, melting bubbles, and S-DNA during DNA overstretching using fluorescence microscopy
Revealing the competition between peeled ssDNA, melting bubbles, and S-DNA during DNA overstretching using fluorescence microscopy
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DOI：
10.1073/pnas.1213676110
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发表时间：
2013-03-05
影响因子：
11.1
通讯作者：
Peterman, Erwin J. G.
中科院分区：
文献类型：
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作者：
King, Graeme A.;Gross, Peter;Peterman, Erwin J. G.
机械应力在许多基因组过程中起着关键作用，例如DNA复制和转录。预测双链（ds）DNA对张力的反应的能力是理解DNA力学的基石。广泛认识到，扭转松弛的dsDNA在类似于65 pN的力下表现出结构转变，称为过度拉伸，由此分子的轮廓长度增加了类似于70%。尽管进行了广泛的研究，但DNA在过度拉伸过程中发生的结构变化仍然引起了相当大的争论。已经提出了三种机制来解释过度拉伸过程中DNA轮廓长度的增加：链解旋，局部碱基对断裂（产生熔融气泡）和S-DNA的形成（链解旋，同时保持碱基配对）。在这里，我们显示，使用荧光显微镜和光镊的组合，这三种结构都可以存在，统一了DNA过度拉伸的往往相互矛盾的教条。我们使用荧光标记蛋白的适当组合来可视化和区分链剥离和熔融气泡形成，而剩余的B型DNA则通过使用特定的荧光分子标记来解释。S-DNA的区域与荧光探针不结合的结构域相关联。我们证明了过度拉伸DNA的三种结构之间的平衡是由DNA拓扑结构和局部DNA稳定性决定的。这些发现增强了我们对DNA力学和稳定性的认识，这对于理解蛋白质如何改变DNA的物理状态至关重要。
Mechanical stress plays a key role in many genomic processes, such as DNA replication and transcription. The ability to predict the response of double-stranded (ds) DNA to tension is a cornerstone of understanding DNA mechanics. It is widely appreciated that torsionally relaxed dsDNA exhibits a structural transition at forces of similar to 65 pN, known as overstretching, whereby the contour length of the molecule increases by similar to 70%. Despite extensive investigation, the structural changes occurring in DNA during overstretching are still generating considerable debate. Three mechanisms have been proposed to account for the increase in DNA contour length during overstretching: strand unpeeling, localized base-pair breaking (yielding melting bubbles), and formation of S-DNA (strand unwinding, while base pairing is maintained). Here we show, using a combination of fluorescence microscopy and optical tweezers, that all three structures can exist, uniting the often contradictory dogmas of DNA overstretching. We visualize and distinguish strand unpeeling and melting-bubble formation using an appropriate combination of fluorescently labeled proteins, whereas remaining B-form DNA is accounted for by using specific fluorescent molecular markers. Regions of S-DNA are associated with domains where fluorescent probes do not bind. We demonstrate that the balance between the three structures of overstretched DNA is governed by both DNA topology and local DNA stability. These findings enhance our knowledge of DNA mechanics and stability, which are of fundamental importance to understanding how proteins modify the physical state of DNA.