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.
