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首页> 外文期刊>Biophysical Journal >spFRET using alternating excitation and FCS reveals progressive DNA unwrapping in nucleosomes.
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spFRET using alternating excitation and FCS reveals progressive DNA unwrapping in nucleosomes.

机译：使用交替激发和FCS的spFRET显示了核小体中进行性DNA的解缠。

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摘要

Accessibility to DNA wrapped in nucleosomes is essential for nuclear processes such as DNA transcription. Large conformational changes in nucleosome structure are required to facilitate protein binding to target sites within nucleosomal DNA. Transient unwrapping of DNA from nucleosome ends can provide an intrinsic exposure of wrapped DNA, allowing proteins to bind DNA that would otherwise be occluded in the nucleosome. The molecular details underlying these mechanisms remain to be resolved. Here we show how DNA unwrapping occurs progressively from both nucleosome ends. We performed single-pair fluorescence resonance energy transfer (spFRET) spectroscopy with alternating laser excitation (ALEX) on nucleosomes either in free solution or confined in a gel after PAGE separation. We combined ALEX-spFRET with a correlation analysis on selected bursts of fluorescence, to resolve a variety of unwrapped nucleosome conformations. The experiments reveal that nucleosomes are unwrapped with an equilibrium constant of approximately 0.2-0.6 at nucleosome ends and approximately 0.1 at a location 27 basepairs inside the nucleosome, but still remain stably associated. Our findings, obtained using a powerful combination of single-molecule fluorescence techniques and gel electrophoresis, emphasize the delicate interplay between DNA accessibility and condensation in chromatin.
机译：对包裹在核小体中的DNA的可及性对于诸如DNA转录之类的核过程至关重要。需要核小体结构中的大构象变化，以促进蛋白质与核小体DNA内的靶位点结合。 DNA从核小体末端的瞬时解缠可提供包裹的DNA的内在暴露，从而使蛋白质与DNA结合，而DNA否则会被核小体阻塞。这些机制背后的分子细节仍有待解决。在这里，我们展示了如何从两个核小体末端逐步发生DNA解缠。我们进行了单对荧光共振能量转移（spFRET）光谱，并在PAGE分离后在游离溶液中或限制在凝胶中对核小体进行了交替激光激发（ALEX）。我们将ALEX-spFRET与所选荧光猝发的相关性分析相结合，以解决各种未包裹的核小体构象。实验表明，核小体在核小体末端具有约0.2-0.6的平衡常数，在核小体内部27个碱基对的位置具有约0.1的平衡常数，但仍保持稳定缔合。我们的发现是通过单分子荧光技术和凝胶电泳的强大结合获得的，强调了DNA可及性与染色质浓缩之间的微妙相互作用。

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