CAREER: Target-Site Search of DNA-Binding Proteins

职业：DNA 结合蛋白的靶位点搜索

• 批准号: 0847050
• 负责人: Andrew Spakowitz
• 金额: $ 57.92万
• 依托单位: Stanford University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0847050&HistoricalAwards=false
• 关键词: CAREER; Target; Site; Search; DNA

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).In this CAREER proposal the PI, proposes to study the target-site search strategy of DNA-binding proteins. The PI will develop a reaction-diffusion methodology to model the facilitated diffusion of a protein finding its target site. The reaction-diffusion theory will capture both the protein dynamics and the conformational behavior of the DNA, building a prediction for protein translocation that can be adapted to different physical models of DNA. The theory will be extended to 2-site binding proteins by combining facilitated diffusion to lock in the first binding site and a subsequent looping mechanism to bind the second site. The PI proposes a two-state mechanism for complex formation in 2-site binding proteins that includes thermal induced looping and a cinching mechanism due to non-specific binding of DNA to the second protein binding site. Analytical theory and Brownian dynamics simulations of supercoiled DNA will be used to develop a theoretical framework to predict the nucleation and growth of supercoils in plasmid DNA, which will be used to address the impact of plectoneme formation and restructuring in the binding kinetics of 1-site and 2-site binding proteins. In conjunction with the theoretical efforts, an integrated experimental program to test the theoretical predictions and guide model development will be developed. Using restriction enzyme digestions as a probe for target-site localization, experiments will be performed to verify the theoretical predictions and the proposed mechanisms for the target-site search. This research proposal represents a multi-faceted approach to understanding how proteins exploit non-specific binding to enhance kinetics, which is central in the biology of small numbers. Outreach efforts include the development and implementation of teaching modules into the education of high school students that are being treated for childhood cancer. The PI will also pair with a high school teacher during the summers to receive feedback on the ease of implementation and student understanding of the teaching modules.

该奖项是根据2009年美国复苏和再投资法案（公法111-5）资助的。在这个职业计划中，PI建议研究DNA结合蛋白的靶点搜索策略。PI将开发一种反应扩散方法，以模拟蛋白质找到其靶位点的促进扩散。反应扩散理论将捕获蛋白质动力学和DNA的构象行为，建立一个可以适应不同DNA物理模型的蛋白质易位预测。该理论将被扩展到2-网站结合蛋白结合的促进扩散，以锁定在第一个结合位点和随后的成环机制，以结合第二个网站。PI提出了2-位点结合蛋白中复合物形成的两态机制，包括热诱导成环和由于DNA与第二蛋白结合位点的非特异性结合而引起的收紧机制。超螺旋DNA的分析理论和布朗动力学模拟将用于开发一个理论框架来预测质粒DNA中超螺旋的成核和生长，这将用于解决1-位点和2-位点结合蛋白的结合动力学中的plectoneme形成和重组的影响。在理论上的努力，一个综合的实验计划，以测试理论预测和指导模型的发展将被开发。使用限制性内切酶digestion作为靶位点定位的探针，将进行实验以验证理论预测和所提出的靶位点搜索机制。这项研究提案代表了一种多方面的方法来理解蛋白质如何利用非特异性结合来增强动力学，这是小数量生物学的核心。外联工作包括在正在接受儿童癌症治疗的高中生的教育中制定和实施教学模块。PI还将在暑假期间与高中教师配对，以获得有关教学模块实施难易程度和学生理解程度的反馈。