Protein Filament Formation in Activating and Modulating Enzymatic DNA Cleavage Specificity

激活和调节 DNA 酶切特异性中的蛋白丝形成

• 批准号: 1410355
• 负责人: Nancy Horton
• 金额: $ 76.23万
• 依托单位: University of Arizona
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1410355&HistoricalAwards=false
• 关键词: Protein; Filament; Formation; Activating; Modulating

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.

在该研究项目中，PI将研究独特的生物技术系统所携带的复杂调节机制。这些投资将为理解这种机制而仅受到广泛赞赏的基础，并预测和操纵其在生物技术应用中的行为。该项目中的研究和相关培训活动将使学术研究界以及生物技术行业受益。该计划将训练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，并成为这个多学科领域的领导者。该项目的结果是，将提供新的实验测量结果，以纳入生物物理学课程中，以允许初级科学家在培训中进行直接的“动手”分析。 PI将继续扩大她的宣传计划，以鼓励和培训来自潜水员的学术和社会背景的科学家。《传统宿主》系统面临着巨大的进化压力，因此，他们已经开发出了非常巧妙的攻击和防御机制。该项目调查了一个如此出色的系统：在Griseus链霉菌中发现的系统。基于其生化活性，Sgrai是一种来自S. griseus的核酸酶，通过与入侵噬菌体DNA结合来激活，只需扩展其DNA序列裂解特异性，并形成可能为宿主DNA而形成的聚合物，以保护宿主DNA，以保护宿主DNA免受其产生的非目标裂解活性。涉及聚合物或细丝形成的酶机制极为罕见，尽管最近的筛选表明这可能比以前想象的更普遍。作为酶调节的潜在新范式，将在该研究项目中研究一些基本问题，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易寡聚的，现在已通过8.6Å的冷冻电子显微镜结构证实。尽管该结构显示了SGRAI二聚体如何在重复的螺旋排列中激活DNA相关的方法，但是基本问题，例如如何激活DNA裂解，如何改变DNA序列特异性以及不交换域（是否在两个DNA限制的晶体结构中发现），因此存在更高的分辨率和更高的分辨率，因此需要更高的分辨率和答案。了解跑步低聚物的功能的功能也很重要，这是确定这种组装的形成如何，其中结合DNA似乎对低聚物稳定性至关重要，加速而不是阻碍多种DNA裂解。最后，已经假设，通过隔离入侵的噬菌体DNA上的sgrai，通过激活Sgrai来保护宿主DNA免受危险的脱靶裂解的作用，可以在保护宿主DNA免受危险的脱靶裂解方面的作用，从而在危险的脱靶裂解中发挥作用。该项目将使用生化和X射线晶体学方法研究跑步低聚物的结构，测量涉及聚合物形成的动力学步骤，并使用稳态的状态荧光方法在反应途径中进行分离，并测试使用体外和体外和in Vage Infection Assays Phage Infection says Phage Polymer的假定生物学作用。

项目成果

The Need for Speed: Run-On Oligomer Filament Formation Provides Maximum Speed with Maximum Sequestration of Activity

• DOI: 10.1128/jvi.01647-18
• 发表时间: 2019-03-01
• 期刊: JOURNAL OF VIROLOGY
• 影响因子: 5.4
• 作者: Barahona, Claudia J.;Basantes, L. Emilia;Horton, N. C.
• 通讯作者: Horton, N. C.

The run-on oligomer filament enzyme mechanism of SgrAI: Part 1. Assembly kinetics of the run-on oligomer filament

• DOI: 10.1074/jbc.ra118.003680
• 发表时间: 2018-09-21
• 期刊: JOURNAL OF BIOLOGICAL CHEMISTRY
• 影响因子: 4.8
• 作者: Park，Chad K.;Sanchez，Jonathan L.;Horton，N. C.
• 通讯作者: Horton，N. C.

The run-on oligomer filament enzyme mechanism of SgrAI: Part 2. Kinetic modeling of the full DNA cleavage pathway

• DOI: 10.1074/jbc.ra118.003682
• 发表时间: 2018-07
• 期刊: The Journal of Biological Chemistry
• 作者: Chad K. Park;Jonathan L. Sanchez;Claudia J. Barahona;L. Basantes;Juan A. Sanchez;Christian Hernandez;N. Horton