De81 chromatography paper

SspI-linearized pMJ5 (100 nM, molecules) and ³²P-labeled triplex-forming oligonucleotide (TFO) (oligo TFO, 22 mer) (100 nM, molecules) were mixed in buffer containing 25 mM MES (pH 5.5), and 10 mM MgCl₂ and incubated for 15 min at 57 °C, followed by cooling to room temperature and overnight incubation. The RAD54 (5 nM) was incubated for 5 min at 20 °C in 100-µl reaction buffer containing 35 mM Tris-HCl, pH 7.2, 3 mM MgCl₂, 100 µg ml⁻¹ BSA, 1 mM DTT, 2 mM ATP, and the ATP-regenerating system (30 U ml⁻¹ creatine phosphokinase and 15 mM creatine phosphate). The reaction was initiated by addition of the triple-helix substrate (pMJ5 + TFO) (0.5 nM, molecules). Overall, 10 µl aliquots were withdrawn at indicated time points and the reaction was quenched by adding 5 µl of stop solution containing 1.36% SDS, 1.4 mg ml⁻¹ proteinase K, 6% glycerol, 0.015% bromophenol blue followed by 15 min incubation at 37 °C. The DNA products were analyzed by electrophoresis in 1.2% agarose gels in buffer containing 40 mM Tris-acetate, pH 5.5, 5 mM sodium acetate, and 1 mM MgCl₂ at 3.5 V cm⁻¹ for 2 h at 4 °C. The gels were dried on DE81 chromatography paper (Whatman) and quantified using a Storm 840 PhosphorImager (GE Healthcare).

The electrophoretic mobility shift assay to determine the DNA-binding capacity of Sae2/pSae2 and the MRX complex was carried out with a 15 μl total reaction volume in a binding buffer containing 25 mM Tris-acetate, pH 7.5, 1 mM dithiothreitol, 2 mM magnesium acetate, 0.1 mg/ml bovine serum albumin (New England Biolabs), and 1 nM (in molecules) DNA substrates. In reactions where MRX was present, 1 mM ATP was added to the buffer. Where indicated, streptavidin (Sigma, 30 nM) was added to the mixture and preincubated for 5 min at room temperature. Purified proteins were then added on ice and incubated for the indicated times at 30 °C. The complexes were mixed with 5 μl loading dye (50% glycerol, 0.1% bromophenol blue), loaded immediately on native polyacrylamide gel (6% or 4%, as indicated, ratio of acrylamide:bisacrylamide 19:1), and separated by electrophoresis in TBE buffer (89 mM Tris base, 89 mM boric acid, 2 mM EDTA) on ice. Gels were dried on DE81 chromatography paper (Whatman), exposed to a phosphor screen, and scanned using a Typhoon Imager (GE Healthcare). Data were quantitated based on the disappearance of the substrate band using the ImageQuant software. Error bars show standard error of the mean. Two-tailed t test was used to calculate p values, where indicated. Differences between two data populations with p values of >0.05 were not considered significant.

PX junctions were prepared by annealing ³²P-labeled forked DNA intermediates (#71/169*, 32 nM) with 3′-tailed DNAs (#170/171, 48 nM) (50% molar excess of cold tailed DNA)^{46 (link)}. The RAD54 or the RAD54 mutant protein (60 nM, unless indicated otherwise) was incubated with ³²P-labeled synthetic PX junction (32 nM, molecules unless indicated otherwise), in a 100-µl BM buffer containing 25 mM Tris-acetate, pH 7.5, 3 mM magnesium acetate, 2 mM ATP, 1 mM dithiothreitol, 100 µg ml⁻¹ BSA, the ATP-regenerating system (30 U ml⁻¹ creatine phosphokinase and 10 mM creatine phosphate). The reactions were carried out at 30 °C. Aliquots (10 µl) were withdrawn at indicated time points, and DNA substrates were deproteinized by treatment with stop solution (1.36% SDS, 1.4 mg ml⁻¹ proteinase K, 6% glycerol, 0.015% bromophenol blue) for 15 min at 37 °C. Samples were analyzed by electrophoresis in 8% polyacrylamide gels (29:1) in 1× TBE buffer (90 mM Tris borate, pH 8.3, and 1 mM EDTA) at room temperature. The gels were dried on DE81 chromatography paper (Whatman) and quantified using a Storm 840 PhosphorImager (GE Healthcare).

The non-mobile ³²P-labeled PX junction (oligos 174/175/176/181) (30 nM, molecules) was incubated in binding buffer containing 25 mM Tris-acetate (pH 7.5), 5 mM magnesium acetate, 100 µg ml⁻¹ BSA, 2 mM DTT, 2 mM ATP, and 10% glycerol for 5 min at 4 °C. Then RAD54 or RAD54 mutants (at indicated concentrations) were added to the reaction mixture followed by additional incubation for 45 min. RAD54–DNA complexes were analyzed by electrophoresis in 6% non-denaturing polyacrylamide gels (29:1) in 0.25× TBE buffer (22.5 mM Tris borate pH 8.3 and 0.5 mM EDTA). The gels were dried on DE81 chromatography paper (Whatman) and the results were quantified using a Storm 840 PhosphorImager (GE Healthcare).
In competition-binding assay, the non-mobile ³²P-labled PX junction (oligos 174/175/176/181) (30 nM, molecules) was incubated with RAD54_1–142 (300 nM) in the presence of varying concentrations of non-radioactive DNA competitors, ssDNA (oligo 2), dsDNA (oligos 1/2), PX junction (oligos 174/175/176/181), X-junction (oligos 174/175/176/177), in the binding buffer for 45 min at 4 °C. The protein–DNA complexes were analyzed by electrophoresis in non-denaturing polyacrylamide gels.

The 70-nt ssDNA (TSO252, S1 Table) was labeled with ³²P using T4 polynucleotide kinase (NEB) and γ-³²P-ATP (Perkin Elmer). Rad52 or its derivatives was incubated with 100 nM of the labeled TSO252 at 37°C for 15 minutes in a 10 μl reaction mixture containing 30 mM TrisAc (pH7.5), 3 mM MgAc, and 20 mM NaCl. The sample was then mixed with 4 μl of TAE loading dye (50% (v/v) glycerol and 0.1% bromophenol blue in Tris-acetate EDTA buffer), loaded onto a 6% polyacrylamide gel (19 cm x 16 cm) in Tris-borate EDTA buffer, and separated by electrophoresis at 200 V. The gel was dried on DE81 chromatography paper (Whatman) and the labeled products were visualized using a BioRad Personal FX phosphor imager. Radioactive signals were quantified using Quantity One software (BioRad), and the percentage of the Rad52-ssDNA complex in reference to total radioactivity in each lane was determined.