High Resolution Single Molecule Study of RecA

RecA在DNA周围形成螺旋丝是同源重组的关键步骤，但这一高度动态的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，可以跟踪RecA长丝的整个生命周期，具有单单体分辨率和毫秒级的时间分辨率。研究表明，大约需要5个RecA单体来成核细丝，细丝在两端生长和收缩，一次一个单体。在接下来的工作中，该小组将开发单分子分析来探索RecA的同源搜索、同源配对和链交换反应。这些分析包括单分子、双色和三色荧光，荧光标记的寡核苷酸的共振能量转移，以及千碱基长度DNA的机械操纵和荧光成像。这些方法是通用的，将用于研究真核生物中的RAD51和古生物中的RADA，并探索单链DNA上RecA微丝的动态性质随序列、pH和ATP浓度的变化。预计这些研究将激发下一波单分子方法在更复杂的生物学问题上的应用。例如，在项目过程中开发的分析方法可以很容易地应用于研究解旋酶介导链的退火反应，并研究基于RNA的调节机制。基于隐马尔可夫模型的无偏见分析工具将进一步完善，并通过在线界面提供给社区。研究将由研究生和本科生进行，在此过程中培养出物理和生物都很流利的新一代年轻科学家。该委员会亦会进一步发展和教授一门名为“物理生物学：从单分子到系统生物学”的研究生程度课程。该奖项由数学及物理科学局的物理部及生物科学局的分子及细胞生物科学部资助。

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.