Ristic, D. et al. Human Rad51 filaments on double- and single-stranded DNA: correlating regular and irregular forms with recombination function. Nucleic Acids Res. 33, 3292-3302

Delft University of Technology, Delft, South Holland, Netherlands
Nucleic Acids Research (Impact Factor: 9.11). 02/2005; 33(10):3292-302. DOI: 10.1093/nar/gki640
Source: PubMed


Recombinase proteins assembled into helical filaments on DNA are believed to be the catalytic core of homologous recombination.
The assembly, disassembly and dynamic rearrangements of this structure must drive the DNA strand exchange reactions of homologous
recombination. The sensitivity of eukaryotic recombinase activity to reaction conditions in vitro suggests that the status of bound nucleotide cofactors is important for function and possibly for filament structure. We
analyzed nucleoprotein filaments formed by the human recombinase Rad51 in a variety of conditions on double-stranded and single-stranded
DNA by scanning force microscopy. Regular filaments with extended double-stranded DNA correlated with active in vitro recombination, possibly due to stabilizing the DNA products of these assays. Though filaments formed readily on single-stranded
DNA, they were very rarely regular structures. The irregular structure of filaments on single-stranded DNA suggests that Rad51
monomers are dynamic in filaments and that regular filaments are transient. Indeed, single molecule force spectroscopy of
Rad51 filament assembly and disassembly in magnetic tweezers revealed protein association and disassociation from many points
along the DNA, with kinetics different from those of RecA. The dynamic rearrangements of proteins and DNA within Rad51 nucleoprotein
filaments could be key events driving strand exchange in homologous recombination.

Download full-text


Available from: John van Noort
  • Source
    • "The ssDNA is held in an extended conformation in the presynaptic filament, being stretched by ∼50% relative to B form duplex DNA, with an axial rise of ∼5.0 Å per nucleotide (6–10). RAD51 has a DNA-stimulated ATPase activity, and although ATP binding is necessary for presynaptic filament assembly, ATP hydrolysis leads to the turnover of RAD51 protomers from DNA (7,11–15). Once assembled, the presynaptic filament engages duplex DNA and conducts a search for homology in the bound duplex. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Homologous recombination catalyzed by the RAD51 recombinase eliminates deleterious DNA lesions from the genome. In the presence of ATP, RAD51 forms a nucleoprotein filament on single-stranded DNA, termed the presynaptic filament, to initiate homologous recombination-mediated DNA double-strand break repair. The SWI5-SFR1 complex stabilizes the presynaptic filament and enhances its ability to mediate the homologous DNA pairing reaction. Here we characterize the RAD51 presynaptic filament stabilization function of the SWI5-SFR1 complex using optical tweezers. Biochemical experiments reveal that SWI5-SFR1 enhances ATP hydrolysis by single-stranded DNA-bound RAD51. Importantly, we show that SWI5-SFR1 acts by facilitating the release of ADP from the presynaptic filament. Our results thus provide mechanistic understanding of the function of SWI5-SFR1 in RAD51-mediated DNA recombination.
    Full-text · Article · Sep 2013 · Nucleic Acids Research
  • Source
    • "The nucleoprotein filaments that catalyze strand exchange are observed qualitatively to be stiff and regular, or less stiff and irregular, largely depending on the status of the bound nucleotide cofactor (17,18). Conditions that favor bound ATP and inhibit ATP hydrolysis correlate with stiff and regular filaments (18). In general, polymers such as recombinase nucleoprotein filaments can be characterized by their bending persistence length (Lp) and torsional stiffness. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Human RAD51 is a key protein in the repair of DNA by homologous recombination. Its assembly onto DNA, which induces changes in DNA structure, results in the formation of a nucleoprotein filament that forms the basis of strand exchange. Here, we determine the structural and mechanical properties of RAD51-dsDNA filaments. Our measurements use two recently developed magnetic tweezers assays, freely orbiting magnetic tweezers and magnetic torque tweezers, designed to measure the twist and torque of individual molecules. By directly monitoring changes in DNA twist on RAD51 binding, we determine the unwinding angle per RAD51 monomer to be 45°, in quantitative agreement with that of its bacterial homolog, RecA. Measurements of the torque that is built up when RAD51-dsDNA filaments are twisted show that under conditions that suppress ATP hydrolysis the torsional persistence length of the RAD51-dsDNA filament exceeds that of its RecA counterpart by a factor of three. Examination of the filament’s torsional stiffness for different combinations of divalent ions and nucleotide cofactors reveals that the Ca2+ ion, apart from suppressing ATPase activity, plays a key role in increasing the torsional stiffness of the filament. These quantitative measurements of RAD51-imposed DNA distortions and accumulated mechanical stress suggest a finely tuned interplay between chemical and mechanical interactions within the RAD51 nucleoprotein filament.
    Full-text · Article · May 2013 · Nucleic Acids Research
  • Source
    • "If presynaptic filament stabilization is the sole function of Swi5–Sfr1, then a presynaptic filament that is rendered stable by another means should be unresponsive to the Swi5–Sfr1 complex. Since the dissociation of Rad51 from ssDNA is intimately linked to ATP hydrolysis (3,12,23–28), one can assemble a stable Rad51 presynaptic filament by using AMP-PNP (Adenylyl-imidodiphosphate), a non-hydrolyzable ATP analog, as nucleotide cofactor. Importantly, little or no significant enhancement of the Rad51-mediated DNA strand exchange reaction occurred when AMP-PNP was used in lieu of ATP (Figure 6A). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Homologous recombination (HR) represents a major error-free pathway to eliminate pre-carcinogenic chromosomal lesions. The DNA strand invasion reaction in HR is mediated by a helical filament of the Rad51 recombinase assembled on single-stranded DNA that is derived from the nucleolytic processing of the primary lesion. Recent studies have found that the human and mouse Swi5 and Sfr1 proteins form a complex that influences Rad51-mediated HR in cells. Here, we provide biophysical evidence that the mouse Swi5–Sfr1 complex has a 1:1 stoichiometry. Importantly, the Swi5–Sfr1 complex, but neither Swi5 nor Sfr1 alone, physically interacts with Rad51 and stimulates Rad51-mediated homologous DNA pairing. This stimulatory effect stems from the stabilization of the Rad51–ssDNA presynaptic filament. Moreover, we provide evidence that the RSfp (rodent Sfr1 proline rich) motif in Sfr1 serves as a negative regulatory element. These results thus reveal an evolutionarily conserved function in the Swi5–Sfr1 complex and furnish valuable information as to the regulatory role of the RSfp motif that isspecific to themammalianSfr1 orthologs.
    Full-text · Article · Apr 2012 · Nucleic Acids Research
Show more