Structure and Function of Holliday Junctions complexed With Proteins Probed by Fluorescence and UV Raman Spectroscopic Methods

荧光和紫外拉曼光谱法探测与蛋白质复合的霍利迪连接体的结构和功能

• 批准号: 0843656
• 负责人: Ishita Mukerji
• 金额: $ 78.5万
• 依托单位: Wesleyan University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0843656&HistoricalAwards=false
• 关键词: Structure; Function; Holliday; Junctions; complexed

This project studies a central intermediate in the important cellular processes of DNA recombination and repair, which rely on the exchange of homologous DNA strands through a four-stranded structure or Holliday junction. Both DNA repair and recombination are vital functions of the cell, which are needed for cell proliferation and maintaining a stable and active genome. Errors in these processes can lead to the growth of tumors and a reduced life span. Protein- and ion-binding leads to a change in junction structure. This study specifically examines how proteins that are known to be involved in repair and recombination and are known to bind to junctions alter these structures. In particular, the ability of these proteins to either facilitate or suppress the exchanging of strands is addressed by studying the structures of the protein-DNA complexes. Two different methods are used to study the interactions: fluorescence spectroscopy and a laser technique, UV resonance Raman spectroscopy. Using the fluorescence method, the structure of the junction and the protein-binding sites are examined and compared. The Raman technique, which examines molecular vibrations, is used to probe the binding site of the ions. By elucidating the nature of these protein-junction complexes and whether they facilitate strand exchange, these studies address the overall mechanism of how DNA recombination occurs in the cell and the overall function of these proteins. This work studies these processes at a basic level and provides a better understanding of the mechanisms of DNA repair and recombination and specifically, the role of these proteins in maintaining a stable genome. Broader impacts that stem from the project are multifold. Teaching and training of undergraduate and graduate students is one of the main impacts of this work. The PI's academic institution is committed to fostering an environment that promotes the integration of research and education at all levels, including the classroom. Several of the courses taught by the PI draw directly from research in her laboratory. The PI actively works to increase the participation of underrepresented groups in science through her activities as chair of the Minority Affairs Committee of the Biophysical Society. These activities include but are not limited to participation in diversity workshops, guest lecturer in a summer mini-course in Biophysics geared towards underrepresented students and acting as a poster judge at conferences for minority students. In addition, the PI is committed to the advancement of women in science and participates in leadership and mentoring activities at the local and national level. The PI also participates in outreach activities such as 'Minds in Motion' at the K-12 level. The majority of these outreach activities are particularly geared towards groups that are traditionally underrepresented in the sciences.

本项目研究了DNA重组和修复的重要细胞过程中的中心中间体，该过程依赖于同源DNA链通过四链结构或Holliday结进行交换。DNA的修复和重组都是细胞的重要功能，是细胞增殖和维持基因组稳定和活性所必需的。这些过程中的错误会导致肿瘤的生长和寿命的缩短。蛋白质和离子结合导致结结构的改变。这项研究专门研究了已知参与修复和重组以及已知与连接结合的蛋白质如何改变这些结构。特别是，这些蛋白质促进或抑制链交换的能力是通过研究蛋白质- dna复合物的结构来解决的。两种不同的方法用于研究相互作用：荧光光谱和激光技术，紫外共振拉曼光谱。利用荧光法，检查和比较了连接和蛋白质结合位点的结构。检测分子振动的拉曼技术被用来探测离子的结合位置。通过阐明这些蛋白质连接复合物的性质以及它们是否促进链交换，这些研究解决了细胞中DNA重组如何发生的总体机制以及这些蛋白质的总体功能。这项工作在基础水平上研究了这些过程，并提供了对DNA修复和重组机制的更好理解，特别是这些蛋白质在维持基因组稳定中的作用。该项目产生的更广泛影响是多方面的。本科生和研究生的教学与培养是这项工作的主要影响之一。PI的学术机构致力于营造一个促进各级研究和教育整合的环境，包括课堂。PI教授的几门课程直接取材于她实验室的研究。PI通过她作为生物物理学会少数民族事务委员会主席的活动，积极致力于增加代表性不足的群体对科学的参与。这些活动包括但不限于参加多样性讲习班，在面向代表性不足的学生的夏季生物物理学迷你课程中担任客座讲师，并在少数民族学生会议上担任海报评委。此外，该计划还致力于提高妇女在科学领域的地位，并参与地方和国家一级的领导和指导活动。PI还参与了诸如K-12级的“运动中的思想”等外展活动。这些外联活动中的大多数特别针对传统上在科学领域代表性不足的群体。