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Mechanistic investigation of processive and distributive DNA modification

进行性和分布性 DNA 修饰的机制研究

基本信息

批准号：
1808775
负责人：
Norbert Reich
金额：
$ 57.75万
依托单位：
University of California-Santa Barbara
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2018
资助国家：
美国
起止时间：
2018-08-01 至 2024-07-31
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1808775&HistoricalAwards=false
关键词：
Mechanistic investigation processive distributive DNA

项目摘要

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Norbert Reich from the University of California at Santa Barbara to understand how proteins scan DNA in order to find specific sites for action. This process is essential to all known cellular systems. A new idea about how this happens is that enhanced motion over large distances requires DNA looping. This highly efficient approach is investigated and modeled using a newly developed theoretical approach. The implications of this work are quite broad as this means of movement is likely to be used by various proteins. This project also supports the development of a university course focused on teaching STEM students how to design experiments through extensive lab work as well as lectures and discussions. Furthermore, the expansion of a large outreach program is proposed (SciTrek) which reaches thousands of K-12 students by bringing trained university students into the classroom for several weeks to guide self-directed investigations on diverse topics. In studying the bacterial DNA adenine methyltransferase (Dam, modifies 5'-GATC-3'), it was noted that the ability to rapidly modify two or more sites is enhanced when the sites are separated up to 500 bp. Furthermore, current models of processive and distributive DNA modification fail to fully account for the efficient search processes by Dam and other proteins. A new understanding of the underlying mechanism is needed. Aim 1 attempts to answer the question: Do proteins relying on different mechanisms display distinct trajectories away from the DNA? Sliding, hopping, intersegmental transfer and intersegmental hopping mechanisms predict distinct protein movements away from the DNA and anovel experimental approach to address this is proposed. The second aim attempts to answer the question: What is the mechanism of intrasite processivity? How widespread is this activity? Dam as well as several other DNA modifying enzymes modifies both strands of DNA within the same recognition site, without dissociating (intrasite processivity). The third aim attempts to answer the question: Is efficient multisite DNA modification important in vivo? The fourth aim develops and applies quantitative theoretical/numerical tools for analyzing activity-based kinetic assays of DNA modifying enzymes. Particular emphasis is placed on elucidating the behavior of Dam, but additional systems (CcrM, uracil DNA glycosylase) are also studied to test and validate the approach more generally.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

有了这个奖项，化学部的生命过程化学计划正在资助圣巴巴拉的加州大学的诺伯特赖希博士，以了解蛋白质如何扫描DNA，以找到特定的作用位点。这个过程对于所有已知的蜂窝系统都是必不可少的。关于这种情况如何发生的一个新想法是，在长距离上增强运动需要DNA循环。这种高效的方法进行了研究和建模使用一个新开发的理论方法。这项工作的意义是相当广泛的，因为这种运动方式可能被各种蛋白质使用。该项目还支持大学课程的开发，重点是通过广泛的实验室工作以及讲座和讨论来教授STEM学生如何设计实验。此外，还提出了一个大型推广计划（SciTrek）的扩展，该计划通过将受过培训的大学生带到教室几周来指导各种主题的自我指导调查，从而达到数千名K-12学生。在研究细菌DNA腺嘌呤甲基转移酶（Dam，修饰5 ′-GATC-3 ′）时，注意到当位点分离至500 bp时，快速修饰两个或多个位点的能力增强。此外，目前的进行性和分配性DNA修饰模型未能完全解释Dam和其他蛋白质的有效搜索过程。需要对潜在机制有新的理解。目的1试图回答这个问题：依赖于不同机制的蛋白质是否显示出远离DNA的不同轨迹？滑动，跳跃，节间转移和节间跳跃机制预测不同的蛋白质运动远离DNA和anovel实验方法来解决这个问题。第二个目标试图回答这个问题：什么是内生过程的机制？这种活动有多普遍？Dam以及其他几种DNA修饰酶在同一识别位点内修饰DNA的两条链，而不解离（位点内持续合成能力）。第三个目标试图回答这个问题：有效的多位点DNA修饰在体内重要吗？第四个目标是开发和应用定量理论/数值工具，用于分析DNA修饰酶的基于活性的动力学测定。特别强调的是阐明大坝的行为，但额外的系统（CcrM，尿嘧啶DNA糖基化酶）也进行了研究，以测试和验证的方法更generally.This奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。