Nucleoprotein structures formed at sites of DNA damage

描述（由申请人提供）：单分子电子显微镜提供了一种强有力的方法来研究蛋白质重塑受损DNA的方式。该应用程序的重点是了解一些中央人类DNA修复和端粒结合蛋白如何在大的，复杂的DNA结构包含损伤，以及它们如何进行修复或信号的存在病变相互作用。这是一个高度互动的项目，代表了与Paul Modrich博士在人类错配因子方面的长期富有成效的合作，Aziz Sancar博士在人类修复信号因子方面的合作，以及Titia deLange博士在端粒结合蛋白方面的合作。在过去的5年里，通过我们自己在这个主题上的工作和这些合作，我们已经发表了20多篇论文。这是一个非常有利的时间来进行这些研究，因为我们已经开发了两个强大的新的EM方法：纳米级的生物指针，提供了一种手段，确定蛋白质的位置内的多蛋白质复合物和甘油喷雾/低电压EM，提供了一个更温和的手段，准备样品的EM。此外，作为这些研究的底物，我们已经产生了含有复制叉或霍利迪路口附近的错配碱基和模型端粒DNA的大型天然DNA。错配修复蛋白的工作将利用Modrich实验室最近在体外重建的缺口定向切除修复。Claspin和Rad 9- Husl-Radl复合物的工作将集中在了解这些蛋白质如何与包含损伤的复制叉相互作用。最近在端粒上发现了离散的多蛋白质复合物，这将有助于对端粒重塑的研究。最后，我们实验室的继续工作将集中在p53作为DNA损伤识别的促进剂。每个系统都提供了一个独特的窗口，可以了解损伤部位和端粒的DNA蛋白质重塑的基本问题，并且从一项研究中获得的信息可以立即应用于其他研究。

DESCRIPTION (provided by applicant): Single molecule electron microscopy provides a powerful approach to study the way in which damaged DNA is remodeled by proteins. The focus of this application is understanding how a number of central human DNA repair and telomere binding proteins interact at large, complex DNA structures containing damage, and how they carry out repair or signal the presence of lesions. This is a highly interactive program which represents longstanding fruitful collaborations with Dr. Paul Modrich working on human mismatch factors, Dr. Aziz Sancar working on human repair signaling factors, and with Dr. Titia deLange working on telomere binding proteins. Together from our own work on this topic and through these collaborations we have published over 20 papers in the past 5 years. This is a highly propitious time to carry out these studies since we have developed two powerful new EM methods: nano-scale biopointers that provide a means of identifying the location of proteins within multi-protein complexes and glycerol spray/low voltage EM that gives a more gentle means of preparing samples for EM. Further, as substrates for these studies, we have produced large natural DNAs containing replication forks or Holliday junctions with nearby mismatched bases and a model telomere DNA. Work on the mismatch repair proteins will take advantage of the recent in vitro reconstitution of nick directed excision repair by the Modrich laboratory. Work on Claspin and the Rad 9- Husl-Radl complex will focus on learning how these proteins interact with replication forks containing damage. Studies of the remodeling of telomeres will take advantage of the recent discovery of discrete multi protein complexes at telomeres. Finally continuing work from our laboratory will focus on p53 as a facilitator of DNA damage recognition. Each system offers a unique window into basic questions of DNA protein remodeling at sites of damage and telomeres and information garnered from one study is immediately applied to the others.