Collaborative Research: Theoretical and Experimental Investigation of Molecular Mechanism of DNA Synaptic Complex Assembly and Dynamics

基因组完整性是定义细胞成功运作、维持和进化的关键特征。细胞中的遗传过程需要特定蛋白质控制的调控区域之间的远距离通信。这种相互作用过程中的错误导致遗传过程的终止，随后是细胞损伤、疾病发展或细胞死亡。这些遗传过程的一个共同特征是由特定蛋白质在两个DNA片段之间瞬时形成复合体。然而，解释蛋白质如何识别特定DNA片段的分子模型仍然不确定。该项目下的理论和实验相结合的研究有望建立一个新的框架，这也将解释如何在长DNA分子中搜索遥远的DNA片段。提出了几种促进科学技术进步与研究生和本科生教育计划相结合的途径。DNA和单分子生物物理学的概念被纳入到UNMC计划的生物物理化学课程中，该课程名为结构生物学和生物物理学研究生培训。这些概念也包括在莱斯大学教授的生物物理化学和化学动力学课程中，以及内布拉斯加-奥马哈大学(UNO)的本科生和UNMC夏季本科生研究计划的全国学者参与的项目。PIs将参加内布拉斯加州青年科学家(YNS)计划，为初中生和高中生提供STEM丰富活动，以及吸引莱斯大学未被充分代表的少数族裔本科生暑期研究计划的活动。这一拟议项目的巨大社会影响将是大学层面的博士后研究员、研究生和本科生的培训。在许多基本的遗传过程中，关键的一步是蛋白质介导的远距离DNA区域之间的突触复合体的组装。突触复合体的形成是基因调控(如Lac抑制子)、位点特异性重组(如FLP、Cre重组酶)和各种基因组整合系统中的普遍现象。然而，关于这些蛋白质在突触体形成过程中如何寻找DNA远端位点的分子机制，人们知之甚少。如果形成突触体所需的特定位置位于同一个DNA分子上，那么对这两个位置的搜索会导致DNA中形成一个环。然而，关于突触体组装的知识非常有限。一项拟议的全面研究计划通过生成一个描述特定位置搜索和突触体组装现象的量化模型来填补这一空白。这个问题是由专门从事实验和理论的实验室进行的协调实验和理论研究来解决的。这个项目背后的中心假设是，滑动和节间转移是搜索两个或更多站点的关键路径；这两条路径之间的划分指定了站点搜索的动态。该项目的目标是描述DNA环和其他定义搜索过程的拓扑特征的作用。本文提出的创新性工作的成功完成将促进对DNA突触体组装所涉及的基本过程的了解，这反过来将解释基因组重排所涉及的机制。所提出的实验和理论方法将为阐明基因组中特定DNA区域的定点重组、整合、切除和倒置等基本遗传过程的机制奠定基础。该项目由生物科学局分子和细胞生物科学部的分子生物物理学和遗传机制集群支持。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Genome integrity is a crucial feature defining the successful functioning of cells, their maintenance, and evolution. The genetic processes in the cell require distant communications between the regulatory regions controlled by specific proteins. Mistakes in this interaction process result in termination of the genetic process followed by cell damage, disease development, or cell death. A common feature of these genetic processes is a transient formation of a complex between two DNA segments by specific proteins. However, a molecular model explaining how the proteins recognize specific DNA segments remains uncertain. The combined theoretical and experimental studies under this project are expected to build a novel framework, which will also explain how the search process of distant DNA segments in a long DNA molecule occurs. Several avenues that facilitate the integration of scientific and technological advances in education programs for graduate and undergraduate students are proposed. The concepts of DNA and single molecule biophysics are incorporated into a Biophysical Chemistry course for the UNMC program 'Graduate training in structural biology and biophysics'. These concepts are also included in Biophysical Chemistry and Chemical Kinetics courses that are taught at Rice University as well the participation in the project of undergraduate students from the University of Nebraska-Omaha (UNO) and nation-wide scholars from the UNMC Summer Undergraduate Research program. The PIs will participate in the Young Nebraska Scientists (YNS) program to provide STEM enrichment activities for middle and high school students and activities to attract the underrepresented minority undergraduate students Summer Research Program at Rice University. A large societal impact from this proposed project will be the training of postdoctoral fellows, graduate students, and undergraduate students at the university level. A critical step in numerous fundamental genetic processes is the protein-mediated assembly of a synaptic complex between distant DNA regions. The formation of a synaptic complex is a general phenomenon found in gene regulation (e.g., Lac repressor), site-specific recombination (e.g., Flp, Cre recombinases), and various genome integration systems. Still, little is known regarding the molecular mechanisms that underlie how such proteins search for DNA distant sites during the formation of synaptosomes. If the specific sites needed for the formation of the synaptosome are located on the same DNA molecule the search for the two sites leads to the formation of a loop in the DNA. However, the knowledge on the assembly of synaptosomes is very limited. A proposed comprehensive research plan fills this gap by generating a quantitative model describing the phenomena of the search of specific sites and the synaptosome assembly. This problem is approached by coordinated experimental and theoretical studies to be conducted by labs that specialize in experiments and theory. The central hypothesis behind this project is that sliding and intersegment transfer are the key pathways in the search for two and more sites; the partition between these two pathways specify the dynamics of the site search. The project objective is to characterize the role of DNA loops and other topological features that define the search process. Successful completion of the innovative work proposed herein will advance knowledge of fundamental processes involved in the DNA synaptosome assembly, which will in turn explain mechanisms involved in the genome rearrangements. The proposed experimental and theoretical approaches will lay a foundation to elucidate mechanisms of such fundamental genetic processes as site-specific recombination, integration, excision, and inversion of specific DNA regions within genomes. This project is supported by the Molecular Biophysics and Genetic Mechanism clusters of the Molecular and Cellular Biosciences Division in the Directorate for Biological Sciences.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.