Structural transitions within human Rad51 nucleoprotein filaments.

Rad51 is a core component of the eukaryotic homologous recombination machinery and is responsible for key mechanistic steps during strand invasion. Higher order oligomers of Rad51 display a remarkable degree of structural variation, forming rings, compressed filaments, and elongated filaments. It is unclear whether Rad51 can transition directly between these different oligomeric structures without disassembling first into monomers.

Visualizing the disassembly of S. cerevisiae Rad51 nucleoprotein filaments.

Rad51 is the core component of the eukaryotic homologous recombination machinery and assembles into elongated nucleoprotein filaments on DNA. We have used total internal reflection fluorescence microscopy and a DNA curtain assay to investigate the dynamics of individual Saccharomyces cerevisiae Rad51 nucleoprotein filaments. For these experiments the DNA molecules were end-labeled with single fluorescent semiconducting nanocrystals.

A DNA-translocating Snf2 molecular motor: Saccharomyces cerevisiae Rdh54 displays processive translocation and extrudes DNA loops.

We have used total internal reflection fluorescence microscopy (TIRFM) to investigate the characteristics of the yeast homologous recombination factor Rdh54 on DNA. Our results demonstrate translocation of Rdh54 on DNA and extrusion of DNA loops by Rdh54 in an ATP hydrolysis-dependent manner. The translocating Rdh54 was highly processive and displayed a variety of behavior, including variations in translocation rate and distance, pauses, and reversals.

Long-distance lateral diffusion of human Rad51 on double-stranded DNA.

Rad51 is the primary eukaryotic recombinase responsible for initiating DNA strand exchange during homologous recombination. Although the subject of intense study for over a decade, many molecular details of the reactions promoted by Rad51 and related recombinases remain unknown. Using total internal reflection fluorescence microscopy, we directly visualized the behavior of individual Rad51 complexes on double-stranded DNA (dsDNA) molecules suspended in an extended configuration above a lipid bilayer.

Simultaneous atomic force microscope and fluorescence measurements of protein unfolding using a calibrated evanescent wave.

Fluorescence techniques for monitoring single-molecule dynamics in the vertical dimension currently do not exist. Here we use an atomic force microscope to calibrate the distance-dependent intensity decay of an evanescent wave. The measured evanescent wave transfer function was then used to convert the vertical motions of a fluorescent particle into displacement (SD = < 1 nm).