Real-Time Observation of RecA Filament Dynamics with Single Monomer Resolution

Abstract

RecA and its homologs help maintain genomic integrity through recombination. Using single-molecule fluorescence assays and hidden Markov modeling, we show the most direct evidence that a RecA filament grows and shrinks primarily one monomer at a time and only at the extremities. Both ends grow and shrink, contrary to expectation, but a higher binding rate at one end is responsible for directional filament growth. Quantitative rate determination also provides insights into how RecA might control DNA accessibility in vivo. We find that about five monomers are sufficient for filament nucleation. Although ordinarily single-stranded DNA binding protein (SSB) prevents filament nucleation, single RecA monomers can easily be added to an existing filament and displace SSB from DNA at the rate of filament extension. This supports the proposal for a passive role of RecA-loading machineries in SSB removal.

Fueling protein–DNA interactions inside porous nanocontainers

“…Upon assembly, RecA filament stretches the ssDNA, pushing the dyes further apart, resulting in weaker dipole interactions between the dyes (low FRET efficiency). RecA monomers dissociate from the ends of the filament upon ATP hydrolysis (19,21), making the filament a highly dynamic structure that can be precisely probed via single-molecule FRET.…”

“…The assembly and disassembly of RecA filament on ssDNA were detected using FRET (19). Here, we approximate the FRET efficiency E by using the ratio between the acceptor intensity and the total intensity.…”

Homebuilt single-molecule scanning confocal fluorescence microscope studies of single DNA/protein interactions

“…A scanning confocal fluorescence microscope (SCFM) that is sensitive enough to detect single fluorescent molecules can be used to observe single-pair fluorescence resonance energy transfer (spFRET) [29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45]. Fluorescence resonance energy transfer (FRET) is a spectroscopic process in which non-radiative energy transfer occurs between an excited dipole (the donor) and another dipole (the acceptor) that has an absorption spectrum that overlaps the emission spectrum of the donor [46].…”

“…Changes in the conformation of the molecule are detected as changes in the energy transfer between the donor and acceptor molecule. In this fashion, single molecular-pair FRET has been used to study, e.g., nanometer conformational motions in individual RNA molecules [29][30][31][32][33][34][35], individual DNA molecules [36][37][38][39][40][41] and individual helicases and other motor proteins on DNA [42][43][44][45]. By following changes in FRET efficiency over time, it is possible to observe nanometer distance changes due to macromolecular rearrangements.…”

Honey, I shrunk the DNA: DNA length as a probe for nucleic‐acid enzyme activity

“…The robustness of DNA as a substrate and the development of techniques to manipulate individual DNA molecules 17 have allowed a large number of nucleic-acid enzymes to be characterized at the single-molecule level. The results of these studies have contributed to new insights in the areas of transcription, [18][19][20][21][22][23] recombination, [24][25][26][27][28][29][30] repair, 31 and replication. [32][33][34][35][36][37] The physical properties of DNA itself have been thoroughly characterized by stretching individual DNA molecules and monitoring their length as a function of parameters such as force and torque.…”