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.

在这个研究项目中，首席研究员将研究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被广泛认识的机制以及预测和操纵其在生物技术应用和相关领域的行为奠定基础。该项目的培训活动将使学术研究界和生物技术行业受益，该计划将培训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，以了解复杂的酶调节机制，成为这个多学科领域的领导者。结果。项目中，新的实验测量将被纳入生物物理课程，以便初级科学家在培训中直接进行“实践”分析。PI 将继续扩大其外展活动，以鼓励和培训科学家。来自不同的学术和社会背景。噬菌体宿主系统面临着巨大的进化压力，因此它们发展出了独特的巧妙的攻击和防御机制，该项目基于其生化特征，研究了一种在灰色链霉菌中发现的非凡系统。活动， SgrAI 是一种来自灰色链球菌的核酸酶，被认为是通过与入侵的噬菌体 DNA 结合而被激活，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 免受其脱靶酶机制的影响。聚合物或丝的形成极其罕见，尽管最近的筛选表明这可能比以前涉及的思想更常见，作为酶调节的潜在新范式，出现了一些基本问题，将在本研究中进行研究。项目，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 形成的聚合物是连续低聚物，现已通过 8.6 Å 冷冻电子显微镜结构得到证实。显示 SgrAI 二聚体如何与重复螺旋排列中的激活 DNA 结合体结合，基本问题例如 DNA 切割如何激活、DNA 序列特异性如何改变以及结构域是否存在交换（在两个 DNA 结合的 SgrAI 二聚体的晶体结构中发现）的存在需要更高的分辨率，因此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方式以及结合的位置。 DNA 对于寡聚体的稳定性至关重要，它会加速而不是阻碍多次 DNA 裂解。最后，连续寡聚体形成的生物学作用已被用来保护宿主 DNA 免受通过激活而可能发生的危险的脱靶裂解。 SgrAI，通过将 SgrAI 隔离在入侵的噬菌体 DNA 上，该项目将使用生化和 X 射线晶体学方法研究连续寡聚物的结构，使用预稳态荧光测量反应途径中涉及聚合物形成和解离的动力学步骤。方法，并使用体外和体内测定（包括噬菌体感染挑战）来测试聚合物的假定生物学作用。

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