Mechanistic investigation of processive and distributive DNA modification

有了这个奖项，化学部门的生命过程化学项目资助了加州大学圣巴巴拉分校的Norbert Reich博士，以了解蛋白质如何扫描DNA以找到特定的作用位点。这个过程对所有已知的细胞系统都是必不可少的。关于这是如何发生的一种新观点是，远距离运动的增强需要DNA环。这种高效的方法是研究和建模使用一个新发展的理论方法。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所使用。该项目还支持开发一门大学课程，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，还提出了一项大型外展计划（SciTrek）的扩展，该计划通过将受过训练的大学生带入课堂，指导他们在几周内对不同主题进行自主调查，从而覆盖数千名K-12学生。在对细菌DNA腺嘌呤甲基转移酶（Dam，修饰5'-GATC-3'）的研究中，我们注意到当两个或多个位点相隔500 bp时，快速修饰两个或多个位点的能力得到增强。此外，目前的过程性和分布式DNA修饰模型未能充分考虑到Dam和其他蛋白质的有效搜索过程。需要对潜在的机制有一个新的认识。Aim 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、节段间转移和节段间跳跃机制预测了不同的蛋白质远离DNA的运动，并提出了新的实验方法来解决这一问题。第二个目标试图回答这个问题：什么是场内加工的机制？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（位点内加工）。第三个目标试图回答这个问题：有效的多位点DNA修饰在体内是否重要？第四个目标是开发和应用定量理论/数值工具来分析DNA修饰酶的基于活性的动力学分析。特别强调的是阐明Dam的行为，但也研究了其他系统（CcrM，尿嘧啶DNA糖基化酶）以更普遍地测试和验证该方法。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.