Visualization of RecA Filaments and DNA by Fluorescence Microscopy

Abstract

We have developed two experimental methods for observing Escherichia coli RecA-DNA filament under a fluorescence microscope. First, RecA-DNA filaments were visualized by immunofluorescence staining with anti-RecA monoclonal antibody. Although the detailed filament structures below submicron scale were unable to be measured accurately due to optical resolution limit, this method has an advantage to analyse a large number of RecA-DNA filaments in a single experiment. Thus, it provides a reliable statistical distribution of the filament morphology. Moreover, not only RecA filament, but also naked DNA region was visualized separately in combination with immunofluorescence staining using anti-DNA monoclonal antibody. Second, by using cysteine derivative RecA protein, RecA-DNA filament was directly labelled by fluorescent reagent, and was able to observe directly under a fluorescence microscope with its enzymatic activity maintained. We showed that the RecA-DNA filament disassembled in the direction from 5 0 to 3 0 of ssDNA as dATP hydrolysis proceeded.

Crystal Structure of the Phage T4 Recombinase UvsX and Its Functional Interaction with the T4 SF2 Helicase UvsW

“…Insights into the transition between the two conformations, and the roles of ATP hydrolysis and DNA tension in controlling this transition have recently been provided by single-molecule studies on Rad5144–46 and RecA 47. Crystallographic analyses of RecA filaments have also revealed how ATP binding at the protomer-protomer interface stabilizes the extended filament in the presence of ssDNA 9.…”

Elastic Behavior of RecA-DNA Helical Filaments

“…In fact, the purified protein expressed activity equivalent to that of the wildtype. 30 To fix the RecA filament on a glass plate, M13mp18 circular ssDNA (7249 bases) was linearized by restriction enzyme, and a biotin molecule was attached at the 3′ terminus of the linear ssDNA via a PEG linker. A 4 μl (3 mm × 18 mm × 0.08 mm) solution exchange chamber was prepared by superposing two cover glasses, and streptavidin, which has four binding sites for biotin, was then immobilized on the glass surface via biotinylated bovine serum albumin (BSA).…”

“…We showed earlier that the major biochemical activities of RecA protein, ATPase and strand exchange activities, were not compromised by the labelling procedure. 30 The fluorescently labelled RecA filament was infused into the flow chamber, and allowed to become immobilized on the glass plate through the biotin-streptavidin binding (Figure 1(b)). Unbound RecA filaments were washed out with solution buffer containing 1 mM dATP and 0.53 μM wildtype RecA.…”

“…The prepared DNA substrate was referred to as M13-linker-biotin ssDNA. The 353C RecA derivative, where one cysteine residue was introduced to the C terminus of a wild-type RecA protein, was prepared and purified as described, 30 and further purified by anion-exchange column chromatography (UNOsphere Q, Bio-Rad). The wild-type RecA protein was purified as described above or was purchased from New England Biolabs (Ipswich, MA, USA).…”

Nanoscale Study of Polymer Dynamics

“…The tube diameter was determined and was shown to decrease with increasing concentration. Further studies on actin filaments and DNA chains have been published in more recent years [21][22][23][24][25][26] . Also a study on the motion of synthetic single-walled carbon nano tubes in a gel, aimed at understanding the effect of flexibility on the diffusion of stiff filaments under confinement, has appeared 27 .…”