Elucidating the Role of Topological Constraint on the Structure of Overstretched DNA Using Fluorescence Polarization Microscopy.
Elucidating the Role of Topological Constraint on the Structure of Overstretched DNA Using Fluorescence Polarization Microscopy.
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DOI：
10.1021/acs.jpcb.1c02708
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发表时间：
2021-08-05
期刊：
影响因子：
--
通讯作者：
Peterman EJG
中科院分区：
文献类型：
--
作者：
Backer AS;King GA;Biebricher AS;Shepherd JW;Noy A;Leake MC;Heller I;Wuite GJL;Peterman EJG
DNA作用力光谱和荧光DNA插层染料的偏光显微镜相结合，可以为研究张力下DNA的结构提供有价值的见解。这些技术以前曾被用来表征S脱氧核糖核酸--当脱氧核糖核酸在≥65 pN的力下过度伸展时形成的一种拉长的脱氧核糖核酸构象。由此推断，S-脱氧核糖核酸的碱基对相对于松弛(B-型)脱氧核糖核酸的碱基对是高度倾斜的。然而，目前尚不清楚DNA上的拓扑约束是否以及如何影响张力下的碱基对倾向性。在这里，我们应用偏光显微镜来研究DNA拉伸几何形状、扭转约束和负超卷曲对超拉伸过程中插层染料取向的影响。与早先的预测相反，拉力几何形状(即DNA分子是通过相反的链还是相同的链拉伸)被发现几乎没有影响。然而，扭转约束导致在过度伸展的DNA中嵌入倾斜的大幅减少，特别是在富含AT的序列中。令人惊讶的是，当DNA分子负超卷曲到临界超卷密度(对应于连接数减少70%的∼)时，嵌入器倾斜的程度也同样减少。我们将这些观察结果归因于P-DNA(一种过度缠绕的DNA构象)的存在。我们的结果表明，插入的DNA优先于P-DNA的侧翼区域，而不是S-DNA的侧翼区域，这也证实了先前的观点，即P-DNA主要形成于富含AT的序列中。
The combination of DNA force spectroscopy and polarization microscopy of fluorescent DNA intercalator dyes can provide valuable insights into the structure of DNA under tension. These techniques have previously been used to characterize S-DNA—an elongated DNA conformation that forms when DNA overstretches at forces ≥ 65 pN. In this way, it was deduced that the base pairs of S-DNA are highly inclined, relative to those in relaxed (B-form) DNA. However, it is unclear whether and how topological constraints on the DNA may influence the base-pair inclinations under tension. Here, we apply polarization microscopy to investigate the impact of DNA pulling geometry, torsional constraint, and negative supercoiling on the orientations of intercalated dyes during overstretching. In contrast to earlier predictions, the pulling geometry (namely, whether the DNA molecule is stretched via opposite strands or the same strand) is found to have little influence. However, torsional constraint leads to a substantial reduction in intercalator tilting in overstretched DNA, particularly in AT-rich sequences. Surprisingly, the extent of intercalator tilting is similarly reduced when the DNA molecule is negatively supercoiled up to a critical supercoiling density (corresponding to ∼70% reduction in the linking number). We attribute these observations to the presence of P-DNA (an overwound DNA conformation). Our results suggest that intercalated DNA preferentially flanks regions of P-DNA rather than those of S-DNA and also substantiate previous suggestions that P-DNA forms predominantly in AT-rich sequences.
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影响因子：
48
作者：
Brockman JM;Blanchard AT;Pui-Yan V Ma;Derricotte WD;Zhang Y;Fay ME;Lam WA;Evangelista FA;Mattheyses AL;Salaita K
通讯作者：
Salaita K
影响因子：
16.6
作者：
Curcio V;Alemán-Castañeda LA;Brown TG;Brasselet S;Alonso MA
通讯作者：
Alonso MA
DOI：
10.1073/pnas.1908826116
发表时间：
2019-12-26
影响因子：
11.1
作者：
King, Graeme A.;Burla, Federica;Wuite, Gijs J. L.
通讯作者：
Wuite, Gijs J. L.
DOI：
10.1364/josab.20.000554
发表时间：
2003-03-01
影响因子：
1.9
作者：
Böhmer, M;Enderlein, J
通讯作者：
Enderlein, J
影响因子：
16.6
作者：
King, Graeme A.;Peterman, Erwin J. G.;Wuite, Gijs J. L.
通讯作者：
Wuite, Gijs J. L.