Nucleoprotein structures formed at sites of DNA damage

DNA 损伤部位形成的核蛋白结构

• 批准号: 7618697
• 负责人: JACK D GRIFFITH
• 金额: $ 32.22万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-26 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7618697
• 关键词: Area; Binding; Binding Proteins; Biochemical; Biological; Collaborations; Communication; Complex; Consensus; Cruciform DNA; DNA; DNA Damage; DNA Folding; DNA Repair; DNA Structure; DNA-Protein Interaction; Dependence; Electron Microscopy; Elements; Event; Excision Repair; Generations; Glycerol; Goals; Hand; Hand functions; Human; Human Papillomavirus; In Vitro; Indium; Individual; Laboratories; Learning; Lesion; Location; Mediator of activation protein; Methods; Mismatch Repair; Modeling; Mus; Nucleoproteins; Paper; Phosphorylation; Polyomavirus; Proteins; Publishing; Replicon; Resolution; Role; Sampling; Signal Transduction; Site; Structure; System; TERF1 gene; TP53 gene; Telomere-Binding Proteins; Testing; Time; Work; base; human DNA; insight; nanoscale; programs; protein complex; reconstitution; repaired; single molecule; success; telomere; voltage

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.

描述（由申请人提供）：单分子电子显微镜提供了一种强大的方法来研究蛋白质重塑受损 DNA 的方式。该应用的重点是了解许多人类核心 DNA 修复和端粒结合蛋白如何在含有损伤的大型复杂 DNA 结构中相互作用，以及它们如何进行修复或发出病变存在的信号。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与 Titia deLange 博士研究端粒结合蛋白的长期富有成果的合作。通过我们自己在这个主题上的工作以及这些合作，我们在过去 5 年里发表了 20 多篇论文。现在是进行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：纳米级生物指针，提供了一种识别多蛋白复合物中蛋白质位置的方法，以及甘油喷雾/低压 EM，可以提供更温和的 EM 样品制备方法。此外，作为这些研究的底物，我们生产了含有复制叉或霍利迪连接以及附近不匹配碱基的大型天然 DNA 和模型端粒 DNA。错配修复蛋白的研究将利用莫德里奇实验室最近对切口定向切除修复的体外重建。 Claspin 和 Rad 9-Husl-Radl 复合物的研究重点是了解这些蛋白质如何与含有损伤的复制叉相互作用。端粒重塑的研究将利用最近在端粒处发现的离散多蛋白复合物。最后，我们实验室的后续工作将重点关注 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来了解损伤部位和端粒处 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。