Protein Filament Formation in Activating and Modulating Enzymatic DNA Cleavage Specificity

在这个研究项目中，PI将研究独特的生物系统所采用的复杂调控机制。这些研究将为理解这些机制奠定基础，这些机制才刚刚得到广泛的重视，并预测和操纵它们的行为，用于生物技术应用。该项目的研究和相关培训活动将使学术研究界和生物技术行业受益。该计划将培养初级科学家开发和实施基于生物物理和生物化学的概念方法，以了解复杂的酶调控机制，成为这一多学科领域的领导者。由于这一项目，将提供新的实验测量，以纳入生物物理课程，使受训的初级科学家能够直接“动手”分析。PI将继续扩大她的推广计划，以鼓励和培训来自不同学术和社会背景的科学家。噬菌体宿主系统处于强烈的进化压力下，因此它们发展出了非常巧妙的攻击和防御机制。本项目研究了一个这样的显着系统：在灰色链霉菌中发现的系统。根据SgrAI的生物化学活性，SgrAI是一种来源于S.据推测，通过与侵入的噬菌体DNA结合，同时扩展其DNA序列切割特异性并形成可用于保护宿主DNA免受其产生的脱靶切割活性的聚合物，从而激活灰毛酵母。涉及聚合物或细丝形成的酶机制非常罕见，尽管最近的筛选表明这可能比以前认为的更常见。作为一个潜在的酶调节的新范式，出现了几个基本问题，将在本研究项目中进行调查，包括结构，动力学和生物作用的聚合物。生物化学数据表明，由活化的SgrAI形成的聚合物是一种连续的寡聚体，现在已经通过8.6 μ m的冷冻电子显微镜结构证实了这一点。虽然这种结构显示了如何结合到激活DNA的SgrAI二聚体在重复的螺旋排列中缔合，但基本问题，如DNA切割如何被激活，DNA序列特异性如何被改变，以及是否存在结构域交换（在两个DNA结合的SgrAI二聚体的晶体结构中发现），需要更高的分辨率，因此仍有待回答。对于理解连续寡聚体的功能也很重要的是确定这种组装体的形成如何加速而不是阻碍多个DNA切割，其中结合的DNA对于寡聚体稳定性似乎是关键的。最后，已经假设连续寡聚体形成的生物学作用在保护宿主DNA免受危险的脱靶切割中起作用，所述脱靶切割通过将SgrAI隔离在入侵的噬菌体DNA上而通过激活SgrAI而成为可能。该项目将使用生物化学和X射线晶体学方法研究连续低聚物的结构，使用预稳态荧光方法测量涉及反应途径中聚合物形成和解离的动力学步骤，并使用体外和体内试验（包括噬菌体感染挑战）测试聚合物的假定生物学作用。

In this research project, the PI will investigate complex regulatory mechanisms employed by unique biological systems. These investigations will build a foundation for understanding such mechanisms that are only just becoming widely appreciated, and for prediction and manipulation of their behavior for biotechnological applications. The research and associated training activities in this project will benefit both the academic research community and as well as the biotechnology industry. The program will train junior scientists to develop and implement biophysical and biochemical based conceptual approaches to understand complex enzyme regulatory mechanisms, to become leaders in this multidisciplinary field. As a result of this project, new experimental measurements will be made available to be incorporated into the biological physics curriculum to allow direct "hands-on" analyses by junior scientists in training. The PI will continue to expand upon her outreach initiatives in an effort to encourage and train scientists from a diverse range of academic and social backgrounds.Phage-host systems are under intense evolutionary pressure, consequently they have developed remarkably ingenious mechanisms of attack and defense. This project investigates one such remarkable system: that found in Streptomyces griseus. Based on its biochemical activities, SgrAI, a nuclease from S. griseus, is postulated to be activated by binding to invading phage DNA, simultaneously expanding its DNA sequence cleavage specificity and forming polymers that may act to protect the host DNA from its resulting off-target cleavage activity. Enzyme mechanisms involving polymer or filament formation are exceedingly rare, although recent screens suggest this may be more common than previously thought. Being a potentially new paradigm for enzyme regulation, several fundamental questions arise that will be investigated in this research project, including the structure, kinetics, and biological role of the polymer. Biochemical data suggests that the polymer formed from activated SgrAI is a run-on oligomer, which has now been confirmed by the 8.6 Å cryo-electron microscopy structure. Although this structure shows how the SgrAI dimers bound to activating DNA associate in a repeating helical arrangement, fundamental questions such as how DNA cleavage is activated, how DNA sequence specificity is altered, and whether or not domain swapping (found in a crystal structure of two DNA bound SgrAI dimers) is present require higher resolution and therefore remain to be answered. Also important to understanding the function of the run-on oligomer is determining how formation of such an assembly, where the bound DNA appears critical for oligomer stability, accelerates rather than impedes multiple DNA cleavages. Finally, the biological role for run-on oligomer formation has been hypothesized to function in protecting the host DNA from dangerous off-target cleavages made possible via activation of SgrAI, by sequestering SgrAI on the invading phage DNA. This project will investigate the structure of the run-on oligomer using biochemical and x-ray crystallographic methods, measure kinetic steps involving polymer formation and dissociation in the reaction pathway using pre-steady state fluorescence methods, and test the postulated biological role of the polymer using in vitro and in vivo assays including phage infection challenges.