Protein Filament Formation in Activating and Modulating Enzymatic DNA Cleavage Specificity

在这个研究项目中，PI将调查独特生物系统所采用的复杂调节机制。这些研究将为理解这些刚刚得到广泛认可的机制，以及为生物技术应用预测和操纵它们的行为奠定基础。这个项目的研究和相关的培训活动将使学术研究界和生物技术工业都受益。该计划将培训初级科学家开发和实施基于生物物理和生化的概念方法，以了解复杂的酶调节机制，成为这个多学科领域的领导者。这个项目的结果是，新的实验测量方法将被纳入生物物理课程，从而允许接受培训的初级科学家直接“动手”分析。PI将继续扩大其推广活动，以鼓励和培训来自不同学术和社会背景的科学家。噬菌体-宿主系统承受着巨大的进化压力，因此它们发展出了非常巧妙的攻击和防御机制。这个项目研究了一个这样非凡的系统：在灰色链霉菌中发现的系统。基于其生化活性，SgrAI，一种来自金黄色葡萄球菌的核酸酶，被假设通过与入侵的噬菌体DNA结合而激活，同时扩大其DNA序列切割特异性并形成聚合物，从而保护宿主DNA免受其脱靶切割活性的影响。参与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。作为酶调控的潜在新范例，本研究项目将研究几个基本问题，包括聚合物的结构、动力学和生物学作用。生化数据表明，由活化的SgrAI形成的聚合物是一种顺行低聚物，目前已被8.6 Å低温电镜结构证实。尽管这种结构显示了SgrAI二聚体如何以重复的螺旋排列结合激活DNA，但诸如DNA切割如何被激活，DNA序列特异性如何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）等基本问题需要更高的分辨率，因此仍有待回答。同样重要的是要了解运行低聚物的功能，确定如何形成这样一个组装，结合的DNA似乎对低聚物的稳定性至关重要，加速而不是阻碍多个DNA切割。最后，通过将SgrAI隔离在入侵的噬菌体DNA上，通过激活SgrAI，可以保护宿主DNA免受危险的脱靶切割。该项目将使用生化和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反应途径中涉及聚合物形成和解离的动力学步骤，并使用体外和体内实验（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.