Single-Molecule Assembly of Protein-DNA Complexes

 描述(由申请人提供)：这项提议的广泛目标是在单分子水平上可视化和量化生物过程的动态行为。我们开发了在单分子水平上研究蛋白质-DNA相互作用的新实验策略。我们设计了几种方法，在这些方法中，我们可以轻松地操纵单个DNA分子，并将其暴露在各种不同的蛋白质、酶和缓冲液中。使用荧光检测，我们可以对DNA上的单个蛋白质进行成像，或者检测它们对单个DNA分子的作用，通常是实时的。我们建议继续使用这种方法来研究重组DNA修复、染色质重塑和DNA复制的未解决的方面。我们寻求继续解决关于RecA/RAD51核蛋白细丝的组装和拆解、它们在同源导向的DNA修复中的控制和功能的悬而未决的问题。此外，我们还建议扩大我们在利用马达蛋白进行染色质重塑方面的兴趣和成功。最后，我们启动了DNA复制的单分子成像研究，并提出了解决与重组DNA修复相交的重要问题。

 DESCRIPTION (provided by applicant): The broad objective of this proposal is to visualize and quantify the dynamic behavior of biological processes at the single-molecule level. We have developed new experimental strategies for studying protein-DNA interactions at the single-molecule level. We devised several approaches wherein we could easily manipulate an individual DNA molecule and expose it to a variety of different proteins, enzymes, and buffers. Using fluorescent detection, we could image individual proteins on DNA or detect their action on individual DNA molecules, typically in real-time. We propose to continue to use this approach to study unresolved aspects of recombinational DNA repair, chromatin remodeling, and DNA replication. We seek to continue to address unresolved questions regarding the assembly and disassembly of RecA/Rad51 nucleoprotein filaments, their control and function in homology-directed DNA repair. In addition, we propose to expand our interests and success in the area of chromatin remodeling by motor proteins. And finally, we have initiated studies on single-molecule imaging of DNA replication, and propose to address important issues that intersect with recombinational DNA repair.