Visualizing the Nonhomogeneous Structure of RAD51 Filaments Using Nanofluidic Channels

Abstract

RAD51 is the key component of the homologous recombination pathway in eukaryotic cells and performs its task by forming filaments on DNA. In this study we investigate the physical properties of RAD51 filaments formed on DNA using nanofluidic channels and fluorescence microscopy. Contrary to the bacterial ortholog RecA, RAD51 forms inhomogeneous filaments on long DNA in vitro, consisting of several protein patches. We demonstrate that a permanent "kink" in the filament is formed where two patches meet if the stretch of naked DNA between the patches is short. The kinks are readily seen in the present microscopy approach but would be hard to identify using conventional single DNA molecule techniques where the DNA is more stretched. We also demonstrate that protein patches separated by longer stretches of bare DNA roll up on each other and this is visualized as transiently overlapping filaments. RAD51 filaments can be formed at several different conditions, varying the cation (Mg 2+ or Ca 2+ ), the DNA substrate (single-stranded or doublestranded), and the RAD51 concentration during filament nucleation, and we compare the properties of the different filaments formed. The results provide important information regarding the physical properties of RAD51 filaments but also demonstrate that nanofluidic channels are perfectly suited to study protein−DNA complexes.

Phosphorylated CtIP bridges DNA to promote annealing of broken ends

“…Efficient bridging also leads to local condensation of DNA along the sugar-phosphate backbone. The formation of circles with local compactions is unique for CtIP and was not observed with, e.g., RAD51, another hallmark HR protein that forms extended filaments in nanofluidic channels (42). Circularization by hybridization was previously observed for the NC chaperone from the HIV-1 virus, but no local compaction was detected (7).…”

Observation of the linewidth broadening of single spins in diamond nanoparticles in aqueous fluid and its relation to the rotational Brownian motion

“…On the nanoscale, the persistent photostability of NV centers, together with the present rotational-motion sensing, will make nanodiamonds indispensable tools to investigate nanoscale fluid mechanics. The classical experimental tool to visualize nanofluids is organic-molecular fluorescent probes 46 , 47 that can also be used for fluorescence depolarization spectroscopy 48 . This method, however, suffers from bleaching of dyes and can only be used for a short period of time and in some specific conditions at specific pH and temperature range.…”

Nanofluidics: A New Arena for Materials Science

“…Because the lipid bilayer exhibited high capability to avoid nonspecific adsorption of proteins (Figure b,c), it enabled the rapid movement of protein–DNA complexes in the nanochannels (Figure d). Therefore, the lipid bilayer‐based coating is very useful to facilitate the direct visualization of the structure and the dynamics of protein–DNA complexes in nanochannels . Last but not the least, it is worthy of attention that more recently Kitamori and co‐workers demonstrated that the use of a nanochannel coated with a lipid bilayer allowed sampling of a single cell content, which has been a challenge in single cell analysis because of the ultrasmall volume.…”