CAREER: Mapping Protein Interactions that Link DNA Replication and Nucleosome Assembly

基因在细胞核中以精确的方式被压缩和组织，以保持非活性基因“关闭”和活性基因“开启”。这些开/关状态的异常变化导致基因组不稳定，并最终影响后代的基因表达。基因表达的维持依赖于几种蛋白质，这些蛋白质在合成后立即一起起作用以浓缩DNA。这项研究旨在了解这些蛋白质的功能和三维结构，以及它们的功能如何协调以维持基因组的非活性区域。该项目的另一个主要目标是为青年科学家创造一个全面的学习环境。大学生将积极参与整个日历年的所有实验，当地高中的学生也将有机会参加每年夏天的研究项目。学生将参加每周的校园科学发展研讨会，并向其他科学家和公众展示他们的研究成果。建议的活动旨在激发这些青年学生对科学的兴趣，并为他们未来的成功职业做好准备。真核生物基因组在DNA复制后立即组织成核小体，这一过程称为复制偶联核小体组装，由两种关键蛋白介导：染色质组装因子1（CAF-1）和增殖细胞核抗原（PCNA）。该项目将采用动力学，热力学和结构方法来了解PCNA如何调节CAF-1活性。等温滴定量热法和表面等离子体共振等技术将用于定量这些蛋白质之间的相互作用，并结合X射线晶体学和小角X射线散射等方法来确定形成的复合物的结构。这些研究的完成将使我们更好地了解DNA复制和核小体组装之间的联系，以及这种调节的中断如何导致异常的基因表达。该项目由生物科学理事会分子和细胞生物科学部的遗传机制计划和刺激竞争研究的既定计划（EPSCoR）共同资助。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Genes are compacted and organized in the nucleus of a cell in a precise manner to keep inactive genes “off” and active genes “on.” Aberrant changes in these on/off states result in genomic instability and ultimately affect gene expression in offspring. Maintenance of gene expression relies on several proteins that function together to condense DNA immediately after it is synthesized. This research aims to understand the function and three-dimensional structure of these proteins and how their functions are coordinated to maintain inactive regions of the genome. Another major goal of this project is to create a comprehensive learning environment for young scientists. College students will be heavily involved in performing all experimentation throughout the calendar year and students from local high schools will also have opportunities to participate in research projects every summer. Students will attend weekly scientific development workshops on campus and present their research results to other scientists and the public. The activities proposed are intended to inspire these young students’ interest in science and prepare them for successful future careers.The eukaryotic genome is organized into nucleosomes immediately following DNA replication in a process called replication-coupled nucleosome assembly, which is mediated by two key proteins: chromatin assembly factor 1 (CAF-1) and proliferating cell nuclear antigen (PCNA). The project will employ kinetic, thermodynamic, and structural approaches to understand how PCNA regulates CAF-1 activity. Techniques such as isothermal titration calorimetry and surface plasmon resonance will be used to quantitate interactions between these proteins, and a combination of methods such as X-ray crystallography and small-angle X-ray scattering will be used to determine the structures of complexes formed. The completion of these studies will lead to a greater understanding of the link between DNA replication and nucleosome assembly and how disruptions in this regulation lead to aberrant gene expression.This project is jointly funded by the Genetic Mechanisms program of the Molecular and Cellular Biosciences Division in the Biological Sciences Directorate and the Established Program to Stimulate Competitive Research (EPSCoR).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.