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. Here we show that complexes of Rad51 can bind to and slide freely along the helical axis of dsDNA. Sliding is bidirectional, does not require ATP hydrolysis, and displays properties consistent with a 1D random walk driven solely by thermal diffusion. The proteins move freely on the DNA for long periods of time; however, sliding terminates and the proteins become immobile upon encountering the free end of a linear dsDNA molecule. This study provides previously uncharacterized insights into the behaviors of human Rad51, which may apply to other members of the RecA-like family of recombinases.DNA repair ͉ homologous recombination ͉ total internal reflection fluorescence microscopy T he repair of double-stranded DNA breaks by homologous recombination is essential for maintaining genome integrity in most organisms (1-3). The importance of homologous recombination is highlighted by the finding that Rad51 null mutations are lethal in mice (4). Furthermore, defects in this repair pathway are associated with a variety of human cancers (5, 6). In eukaryotes, the broken ends of chromosomes are processed by 5Ј to 3Ј exonucleases to yield long single-stranded DNA overhangs (2, 3). Rad51, a DNA-dependent ATPase, assembles onto these overhangs, forming a nucleoprotein filament that is a key intermediate in homologous recombination (1,2,7,8). The primary functions of this filament are to locate homologous sequence that can be used as a template to repair the damaged DNA strand and to initiate strand exchange (1, 7).The structure and function of the complexes formed by Rad51 and the other RecA-like recombinases are conserved throughout evolution (8, 9). In their active states, Rad51 and related recombinases form a helical filament on DNA that induces a 50% extension of the bound DNA molecule (8). The extended nucleoprotein filament is correlated with DNA recombination activity; however, these proteins also form inactive filaments with shortened pitches (Ϸ65-85 Å versus Ϸ90-130 Å) (10). Rad51 and related recombinases also form octameric rings with a central pore large enough to accommodate a double-stranded (dsDNA) molecule (11-16). These ring-like recombinase structures do not appear to be the form of the protein that is active during the strand exchange phase of homologous recombination. Although the biological role of these rings remains unknown, it has been suggested that they may function as DNA ''pumps,'' allowing the proteins to move along DNA (12, 13).Here we have developed a unique total internal reflection fluorescence micr...

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 different behaviors, including variations in translocation rate and distance, pauses, and reversals. We provide evidence that The DNA loops generated encompass an average of six kilobases and Rdh54 often abruptly releases the extruded DNA. Rdh54 forms a multimeric complex which we speculate has at least two functionally distinct DNA-binding sites, one of which enables translocation while the other remains anchored to another DNA locale. Our work, together with other recent studies, suggests that translocation-coupled DNA loop extrusion is a common mechanistic feature among the Snf2-family of chromatin-remodeling proteins.

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). We demonstrate the use of the calibrated evanescent wave to resolve the 20.1 ؎ 0.5-nm step increases in the length of the small protein ubiquitin during forced unfolding. The experiments that we report here make an important contribution to fluorescence microscopy by demonstrating the unambiguous optical tracking of a single molecule with a resolution comparable to that of an atomic force microscope.