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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Dividing a supercoiled DNA molecule into two independent topological domains
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Dividing a supercoiled DNA molecule into two independent topological domains

机译：将超螺旋DNA分子分为两个独立的拓扑域

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摘要

Both prokaryotic and eukaryotic chromosomes are organized into many independent topological domains. These topological domains may be formed through constraining each DNA end from rotating by interacting with nuclear proteins; i.e., DNA-binding proteins. However, so far, evidence to support this hypothesis is still elusive. Here we developed two biochemical methods; i.e., DNA-nicking and DNA-gyrase methods to examine whether certain sequence-specific DNA-binding proteins are capable of separating a supercoiled DNA molecule into distinct topological domains. Our approach is based on the successful construction of a series of plasmid DNA templates that contain many tandem copies of one or two DNA-binding sites in two different locations. With these approaches and atomic force microscopy, we discovered that several sequence-specific DNA-binding proteins; i.e., lac repressor, gal repressor, and λ O protein, are able to divide a supercoiled DNA molecule into two independent topological domains. These topological domains are stable under our experimental conditions. Our results can be explained by a topological barrier model in which nucleoprotein complexes confine DNA supercoils to localized regions. We propose that DNA topological barriers are certain nucleoprotein complexes that contain stable toroidal supercoils assembled from DNA-looping or tightly wrapping DNA around DNA-binding proteins. The DNA topological barrier model may be a general mechanism for certain DNA-binding proteins, such as histone or histone-like proteins, to modulate topology of chromosome DNA in vivo.
机译：原核和真核染色体都组织成许多独立的拓扑结构域。这些拓扑结构域可以通过限制每个DNA末端与核蛋白相互作用而旋转而形成；即DNA结合蛋白。但是，到目前为止，支持这一假设的证据仍然难以捉摸。在这里，我们开发了两种生化方法；即DNA标记法和DNA旋转酶法，以检查某些特定于序列的DNA结合蛋白是否能够将超螺旋DNA分子分离为不同的拓扑结构域。我们的方法基于成功构建一系列质粒DNA模板，其中包含在两个不同位置的一个或两个DNA结合位点的许多串联拷贝。通过这些方法和原子力显微镜，我们发现了几种特定于序列的DNA结合蛋白。即lac阻遏物，gal阻遏物和λO蛋白能够将超螺旋DNA分子分为两个独立的拓扑结构域。这些拓扑结构域在我们的实验条件下是稳定的。我们的结果可以通过拓扑屏障模型来解释，其中核蛋白复合物将DNA超螺旋限制在局部区域。我们提出，DNA拓扑障碍是某些核蛋白复合物，其中包含稳定的环形超螺旋，该环形超螺旋由DNA循环或将DNA紧紧包裹在DNA结合蛋白周围组装而成。 DNA拓扑屏障模型可能是某些DNA结合蛋白（例如组蛋白或类蛋白蛋白）在体内调节染色体DNA拓扑结构的一般机制。

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    Fenfei Leng; Bo Chen; David D. Dunlap;

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    Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th Street, Miami, FL 33199;

    Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th Street, Miami, FL 33199;

    Department of Cell Biology,Emory University, Atlanta, GA 30322;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
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