High Resolution Single Molecule Study of RecA

RecA 的高分辨率单分子研究

• 批准号: 0646550
• 负责人: Taekjip Ha
• 金额: $ 30.33万
• 依托单位: University of Illinois at Urbana-Champaign
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0646550&HistoricalAwards=false
• 关键词: Resolution; Single; Molecule; Study; RecA

The formation of a helical filament around DNA by RecA is a key step in homologous recombination, but the details of this highly dynamic process have remained enigmatic. In earlier work the PI developed a single molecule fluorescence approach that can follow the entire lifecycle of the RecA filament with the single monomer resolution and milliseconds time resolution. Studies showed that about 5 RecA monomers are needed to nucleate the filament and the filament grows and shrinks at both ends, and one monomer at a time. In this continuation the group will develop single molecule assays to probe the homology search, homologous pairing and strand exchange reactions by RecA. These assays include single molecule two-color and three-color fluorescence resonance energy transfer on fluorescently labeled oligonucleotides and mechanical manipulation and fluorescence imaging of kilobase length DNA. The approaches are general and will be used to study the less understood relatives, Rad51 in Eukaryotes and RadA in Archaea, and to probe the dynamic nature of the RecA filament on single stranded DNA as a function of sequence, pH and ATP concentration. It is expected that these studies will inspire the next wave of applications of single molecule approaches to more complex biological problems. For example, the assays developed in the course of the project can be readily applied to study helicase-mediate strand annealing reactions and to investigate the mechanism of RNA-based regulation. The unbiased analytical tool based on Hidden Markov modeling will be further refined and made available to the community via online interface. Research will be performed by graduate students and undergraduate students, in the process training a new generation of young scientists who are fluent both in physics and in biology. The PI will also further develop and teach a graduate level course titled 'Physical Biology: From single molecules to systems biology'.This award is funded by the Division of Physics in the Directorate for Mathematical and Physical Sciences and by the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences.

RecA 在 DNA 周围形成螺旋丝是同源重组的关键步骤，但这一高度动态过程的细节仍然是个谜。在早期的工作中，PI 开发了一种单分子荧光方法，可以以单单体分辨率和毫秒时间分辨率跟踪 RecA 灯丝的整个生命周期。研究表明，大约需要 5 个 RecA 单体才能使细丝成核，并且细丝在两端生长和收缩，每次需要一个单体。 在此延续中，该小组将开发单分子测定法，以探索 RecA 的同源搜索、同源配对和链交换反应。这些测定包括荧光标记寡核苷酸上的单分子二色和三色荧光共振能量转移以及千碱基长度 DNA 的机械操作和荧光成像。这些方法是通用的，将用于研究不太了解的亲属，真核生物中的 Rad51 和古细菌中的 RadA，并探测单链 DNA 上的 RecA 丝作为序列、pH 和 ATP 浓度的函数的动态性质。预计这些研究将激发下一波单分子方法在更复杂的生物问题上的应用。例如，该项目过程中开发的检测方法可轻松应用于研究解旋酶介导的链退火反应并研究基于 RNA 的调控机制。基于隐马尔可夫模型的无偏分析工具将进一步完善，并通过在线界面向社区提供。研究将由研究生和本科生进行，在此过程中培养精通物理学和生物学的新一代年轻科学家。 PI还将进一步开发和教授名为“物理生物学：从单分子到系统生物学”的研究生课程。该奖项由数学和物理科学理事会物理司以及生物科学理事会分子和细胞生物科学司资助。