Nucleoprotein structures formed at sites of DNA damage

描述(申请人提供)：单分子电子显微镜为研究蛋白质重塑受损DNA的方式提供了一种强有力的方法。这一应用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包含损伤的复杂DNA结构中相互作用，以及它们如何进行修复或发出损伤存在的信号。这是一个高度互动的项目，代表了与Paul Modrich博士研究人类错配因子、Aziz Sancar博士研究人类修复信号因子以及Titia DeLange博士研究端粒结合蛋白的长期卓有成效的合作。在过去的5年里，通过我们自己在这个主题上的工作以及通过这些合作，我们发表了20多篇论文。现在是开展这些研究的非常有利的时机，因为我们已经开发了两种强大的新EM方法：纳米级生物探针，它提供了一种识别多蛋白质复合体中蛋白质位置的方法，以及甘油喷雾/低电压EM，它提供了一种更温和的方法来制备EM样品。此外，作为这些研究的底物，我们已经生产了包含复制叉子或与附近不匹配碱基的Holliday连接的大的天然DNA和一个模型端粒DNA。错配修复蛋白的研究将利用莫德里奇实验室最近对缺口定向切除修复进行的体外重组。对Claspin和Rad 9-Husl-Radl复合体的研究将集中在了解这些蛋白质如何与含有损伤的复制叉子相互作用。端粒重塑的研究将利用最近发现的端粒上离散的多蛋白质复合体。最后，我们实验室将继续致力于研究P53作为DNA损伤识别的促进剂。每个系统都为了解损伤部位和端粒DNA蛋白质重塑的基本问题提供了一个独特的窗口，从一个研究中获得的信息立即应用到其他研究中。

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.