Superhelix organization by DNA curvature as measured through site-specific labeling
Superhelix organization by DNA curvature as measured through site-specific labeling
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DOI：
10.1006/jmbi.1997.1476
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发表时间：
1998-01-30
影响因子：
5.6
通讯作者：
Langowski, J
中科院分区：
文献类型：
--
作者：
Pfannschmidt, C;Langowski, J
为了确定超螺旋 DNA 中特定位点的位置，我们开发了一种在特定序列处引入共价生物素标签同时保留超螺旋性的方法。这是通过首先引入特定切口、通过有限切口平移标记 DNA 并用连接酶密封切口来完成的。通过在连接反应中加入乙锭来控制超螺旋性。然后，我们使用显微镜扫描力对用这种方法标记的 DNA 进行扫描，比较了特定位点上链霉亲和素标记相对于超螺旋末端环的位置。我们发现，在具有永久弯曲插入片段的 DNA 中，标签优先位于距末端环一定距离处，而在没有弯曲插入片段的对照中，标签位置是随机的。这表明曲线位于超螺旋的末端环中或附近。 (C) 1998 学术出版社有限公司。
For determining the position of a defined site in a superhelical DNA we have developed a method for introducing a covalent biotin label at a specific sequence while preserving the superhelicity. This is done by first introducing a specific nick, labeling the DNA by Limited nick translation and sealing the nick with ligase. The superhelicity is controlled by including ethidium in the ligation reaction.Using scanning force of microscopy on DNAs labeled by this method, we have then compared the position of streptavidin markers at a specific site relative to the end loop of the superhelix. We found that in DNAs with permanently curved inserts the label is located preferentially at a defined distance from the end loop, while in controls without curved inserts the label position was random. This indicates that curves are located in or near the end loops in a superhelix. (C) 1998 Academic Press Limited.