Purification and characterization of Rad51 proteins. (A) Saccharomyces cerevisiae Rad51 (1 µg, lane 1), Rad51-K191R (1 µg, lane 2) and Rad51-K191A (1 µg, lane 3) proteins were purified from budding yeast, analyzed by 10% SDS–PAGE and stained with Coomassie brilliant blue R250. (B) ATPase activity of Rad51 (dark bars), Rad51-K191R (gray bars) and Rad51-K191A (white bars) proteins. The ATPase activities of Rad51 proteins (2.5 µM) were measured either in the presence of ssDNA (30 µM, poly-dA) or absence of DNA by an NADH-coupled microplate photometric assay. ATPase activity is measured as the rate of NADH decomposition (molar molecules of NADH decomposed per minute by molar molecule of Rad51). All measurements were done in triplicate, and the error bars represent 1 SD. (C) α-³²P-ATP binding by Rad51 proteins. The Rad51-ATP complexes were resolved by 10% SDS–PAGE, and the signal intensities were quantified with a PhosphoImager. The intensity of the Rad51 band at 1 µM ATP was defined as one unit, and the signals were normalized to this standard. (D) Data obtained from (C) and additional gels were fitted into a saturation-binding curves using PRISM software (Graphpad). (E) α-P³²-8-Azido-ATP binding by Rad51 proteins. (F) Fitted saturation binding curves for data obtained from (E) and additional gels. Procedures were as in (C, D). For (C–F), the calculated K_d values are shown in Table 1. All measurements were done in triplicate and the error bars represent 1 SD. For (C, D), the positions of MW markers, BSA and the Rad51 proteins were determined by Coomassie staining and are indicated on the left and right sides.

Rad51 and Rad51-K191R-dsDNA filaments modulate the Rad54 ATPase activity. ATPase assays were performed by incubating 3.3 nM Rad54 together with the Rad51 or Rad51-K191R-dsDNA filaments (6 µM bp) formed either at sub-saturated ratio of 1/37 bp (A), or at saturated ratio of 1/3 bp (B). For comparison, the Rad54 ATPase was also measured on protein-free dsDNA. ATPase reactions were performed under standard buffer conditions at 30°C. ATPase data were fitted into the Michaelis–Menten equation using PRISM software (Graphpad). Data points represent the average of at least three replicate experiments, and the error bars represent 1 SD. The K_M and k_cat values calculated for these curves are shown in Table 3.

Topological assay of Rad51-dsDNA filament disassembly by Rad54. (A) Scheme of the assay. Nucleoprotein filaments were formed by incubating Rad51 or Rad51-K191R protein with the supercoiled DNA substrate. Binding by Rad51 unwinds the dsDNA and leads to the introduction of compensatory positive scs (+). Relaxation of these scs by topoisomerase I followed by deproteinization results in highly negatively supercoiled DNA, so called form X DNA (x, top). If Rad51 is completely removed from the DNA substrate by Rad54 before the treatment of topoisomerase, the substrate will be converted to a fully relaxed species (re, bottom). If Rad51 was partially removed, it will be converted to species with varying degree of negative supercoiling (x, top; sc, middle). (B) ATPase activity of Rad51 is important for its dissociation from dsDNA promoted by Rad54. Rad51 or Rad51-K191R (7.5 µM) was pre-incubated with pUC19 dsDNA (30 µM) for 30 min at 23°C to form nucleoprotein filaments. Rad54 (0.375 µM) was added and incubated for 5 min, followed by the addition of scavenger DNA (63 µM PstI-linearized M13mp19 dsDNA), and incubated for 2 h at 23°C. Reactions were separated on a native 1.2% agarose gel without chloroquine in the first dimension followed by a second electrophoresis step with 4 µg/ml chloroquine. The positions of supercoiled (sc), relaxed (re), X form (x) and scavenger (scav) dsDNA are indicated in the first gel for the reaction with Rad51 protein.

Rad51-DNA filament formation and stability. (A) Binding of Rad51 and Rad51-K191R proteins to dsDNA. Protein titration reactions were performed in ATPase reaction buffer by incubating the 600-bp DNA substrate (6 µM) with various amounts of protein (0.1, 0.2, 0.5, 1, 1.5, 2, 3, 6 µM) at 30°C for 15 min. Protein-free, labeled DNA substrate (lanes 1 and 18); Rad51 (lanes 2–9); Rad51-K191R (lanes 10–17). (B) Salt titration of Rad51-ssDNA and Rad51-K191R-ssDNA complex formation. The nucleoprotein complexes were assembled by incubating 2 µM of Rad51 (lanes 2–10), or Rad51-K191R (lanes 11–19) together with 6-µM ssDNA (621mer) in presence of the indicated sodium chloride concentration for 15 min at 30°C. Protein-free, labeled DNA substrate (lanes 1 and 20). (C, D) Quantification of the results of the Rad51 and Rad51-K191R-DNA complex formation and stability with ssDNA (C) and dsDNA (D). The data were fitted into a sigmoidal curve by PRISM software (GraphPad). All measurements were done in triplicate and the error bars represent 1 SD. The STM data calculated for these curves are shown in Table 2. (E) Electron micrographs of Rad51-DNA filaments formed by wild-type Rad51 (left) and Rad51-K191R mutant proteins (right). The filaments are formed on linear dsDNA in the presence of ATP. The scale bar is 500 Å.

Both Rad51 and Rad54 ATPase activities are important for Rad51-dsDNA filament disassembly. Rad51-dsDNA filaments were formed at 4 bp/Rad51 ratio (1.5 μM Rad51, 6 μM dsDNA) for 15 min at 30°C. Initiation of Rad51-dsDNA filament disassembly was started by the addition of Rad54 (0.1 µM) and scavenger DNA (30 µM). (A) Protein-free, labeled DNA substrate (lane 1); Rad51-dsDNA filaments in the absence of Rad54 but with scavenger DNA (lanes 2–5), in the presence of Rad54 and scavenger DNA (lanes 6–9), in the presence of Rad54 but absence of scavenger DNA (lanes 10–13), in the absence of Rad54 and scavenger DNA (lanes 14–17). (B) The data from (A) and from assays where Rad54-K341R was used instead of wild-type Rad54 protein (data not shown) were quantified on a PhosphoImager. The accumulation of the band of free DNA and the reduction of lowest mobility species of the nucleoprotein filament (saturated complex) were taken as the readout of Rad51 displacement activity by Rad54, respectively. Data points represent the average of at least three replicate experiments, and error bars represent 1 SD. For the reactions with Rad51 and Rad51+Rad54 only the 90 min data are shown. (C) Rad51-K191R-dsDNA filaments were formed at 4 bp/Rad51-K191R ratio (1.5 μM Rad51-K191R, 6 μM dsDNA) for 15 min at 30°C. Initiation of Rad51-K191R-dsDNA filament disassembly was started by the addition of Rad54 (0.1 µM) and scavenger DNA (30 µM). Protein-free, labeled DNA substrate (lane 1); Rad51-K191R-dsDNA filaments in the absence of Rad54 but with scavenger DNA (lanes 2–5), in the presence of Rad54 and scavenger DNA (lanes 6–9), in the presence of Rad54 but absence of scavenger DNA (lanes 10–13), in the absence of Rad54 and scavenger DNA (lanes 14–17). (D) The data from (C) were quantified on a PhosphoImager. The reduction of the saturated nucleoprotein filament was taken as readout of Rad51-K191R displacement by Rad54. Data points represent the average of at least three replicate experiments, and error bars represent 1 SD. For the reactions with Rad51-K191R, Rad51-K191R+Rad54 and Rad51-K191R+Rad54-K341R+scavenger DNA only the 90 min data are shown.

The effects of nucleotide cofactor on Rad51-dsDNA filament disassembly promoted by Rad54. (A) Rad51-dsDNA filaments were assembled at 4 bp/Rad51 (1.5 μM Rad51, 6 μM dsDNA) for 15 min at 30°C, either in the presence of 80% ADP (1 mM ATP and 4 mM ADP) or absence of ADP (5 mM ATP). Addition of Rad54 (50 nM) and scavenger DNA (30 µM) initiated the protein–DNA filament disassembly. Protein-free, labeled DNA substrate (lane 1); Rad51-dsDNA filaments in the absence of Rad54 but with ADP (lanes 2–5), in the presence of Rad54 and ADP (lanes 6–9), in the absence of Rad54 and ADP (lanes 10–13), in the presence of Rad54 but absence of ADP (lanes 14–17). (B) Rad51-dsDNA filaments were assembled as in (A) either in the presence of 20% ADP (4 mM ATP and 1 mM ADP) or absence of ADP (5 mM ATP). Addition of Rad54 (50 nM) and scavenger DNA (30 µM) initiated the protein–DNA filament disassembly. Protein-free, labeled DNA substrate (lane 1); Rad51-dsDNA filaments in the absence of Rad54 but with ADP (lanes 2–5), in the presence of Rad54 and ADP (lanes 6–9), in the absence of Rad54 and ADP (lanes 10–13), in the presence of Rad54 but absence of ADP (lanes 14–17). (C) The data from (A) and (B) were quantified on a PhosphoImager. The accumulation of free DNA was taken as readout of Rad51 displacement activity by Rad54. Data points represent the average of at least three replicate experiments and error bars represent one standard deviation. (D) Rad51-dsDNA filaments were assembled as in (A), either in the presence of ATP-γ-S (50 µM) or ATP (50 µM). Addition of Rad54 (50 nM), scavenger DNA (30 µM) and ATP (5 mM) initiates the protein–DNA filament disassembly. Protein-free, labeled DNA substrate (lane 1); Rad51-dsDNA filaments in the presence of Rad54 and ATP-γ-S (lanes 2–5), in the absence of Rad54 but with ATP-γ-S (lanes 6–9), in the presence of Rad54 but absence of ATP-γ-S (lanes 10–13), in the absence of Rad54 or ATP-γ-S (lanes 14–17). (E) The data from (D) were quantified on a PhosphoImager. The accumulation of the band of free DNA was taken as readout of Rad51 displacement activity by Rad54. Data points represent the average of at least three replicate experiments, and error bars represent 1 SD.