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

Abstract

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...

On the influence of protein-DNA register during homologous recombination

“…2C). One-dimensional diffusion of proteins on both ssDNA and dsDNA is now a well-documented phenomenon, [23][24][25][26][27][28][29][30][31] and we have previously observed 1D diffusion of human Rad51 along dsDNA, 32 supporting the notion that Rad51/RecA monomers within the presynaptic complex might also be capable of sliding at least short distances along ssDNA. The ability of Rad51/RecA to slide just §1-nucleotide would ensure optimal alignment of DNA triplet sequences during the early stages of homology recognition and strand exchange.…”

Fluidic supramolecular nano- and microfibres as molecular rails for regulated movement of nanosubstances

“…Very recently, an increasing number of microtubule-binding proteins have been found to exhibit diff usional motility along the microtubule lattice (one-dimensional random walk) 20,21 , which did not require ATP and is thus regarded as an economical mode of transport. Similar one-dimensional diff usive motion without ATP hydrolysis was observed for a DNA-binding protein moving along the DNA fi bre 22,23 . However, the proportion and importance of the diff usive component contribution into the one-dimensional transport is still controversial.…”

The importance of surfaces in single-molecule bioscience

“…This can be accomplished by tethering the DNA to the surface at just one end (typically via a biotin-streptavidin linkage), in which case buffer flow is necessary to stretch the molecules, or by anchoring both ends in an extended configuration along the surface, which does not require continuous buffer flow. 92,93 Using TIRFM and λ DNA our laboratory has studied the behaviors of DNA damage repair proteins such as the Rad51 recombinase, mismatch recognition proteins, and chromatin remodeling enzymes. 33,[92][93][94] …”

“…92,93 Using TIRFM and λ DNA our laboratory has studied the behaviors of DNA damage repair proteins such as the Rad51 recombinase, mismatch recognition proteins, and chromatin remodeling enzymes. 33,[92][93][94] …”