Journal: Nucleic Acids Research

Article Title: Rad51 and Rad54 ATPase activities are both required to modulate Rad51-dsDNA filament dynamics

doi: 10.1093/nar/gkm412

Figure Lengend Snippet: 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- 32 P-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-P 32 -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 . 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.

Article Snippet: The data were fitted into a sigmoidal curve by PRISM software (GraphPad).

Techniques: Purification, SDS Page, Staining, Activity Assay, Binding Assay, Software

Journal: Nucleic Acids Research

Article Title: Rad51 and Rad54 ATPase activities are both required to modulate Rad51-dsDNA filament dynamics

doi: 10.1093/nar/gkm412

Figure Lengend Snippet: 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 . ( 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.

Article Snippet: The data were fitted into a sigmoidal curve by PRISM software (GraphPad).

Techniques: Binding Assay, Titration, Labeling, Concentration Assay, Software, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Rad51 and Rad54 ATPase activities are both required to modulate Rad51-dsDNA filament dynamics

doi: 10.1093/nar/gkm412

Figure Lengend Snippet: 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 .

Article Snippet: The data were fitted into a sigmoidal curve by PRISM software (GraphPad).

Techniques: Activity Assay, Comparison, Software