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. Author manuscript; available in PMC: 2023 Aug 17.
Published in final edited form as: Methods Mol Biol. 2021;2153:101–113. doi: 10.1007/978-1-0716-0644-5_8

DNA Strand Exchange to Monitor Human RAD51-Mediated Strand Invasion and Pairing

Sudipta Lahiri 1, Ryan B Jensen 1
PMCID: PMC10434838  NIHMSID: NIHMS1920455  PMID: 32840775

Abstract

The homologous recombination (HR) pathway maintains genomic integrity by repairing DNA double-strand breaks (DSBs), single-strand DNA gaps, and collapsed replication forks. The process of HR involves strand invasion, homology search, and DNA strand exchange between paired DNA molecules. HR is critical for the high-fidelity repair of DNA DSBs in mitotic cells and for the exchange of genetic information during meiosis. Here we describe a DNA strand exchange reaction in vitro utilizing purified proteins and defined DNA substrates to measure the strand invasion and pairing activities of the human RAD51 protein. We further discuss how this reaction can be catalytically stimulated by the mediator protein BRCA2.

Keywords: RAD51, BRCA2, RPA, DNA double-strand breaks, Strand invasion, DNA strand exchange, Homologous recombination, Genomic instability

1. Introduction

Homologous recombination (HR) is canonically associated with the exchange of genetic information between identical or highly similar DNA sequences [1]. In human mitotic cells, DNA double-strand breaks (DSBs) that arise from DNA damage can be repaired by recombination between sister chromatids preserving genome integrity [2]. In meiosis, however, programmed DNA DSBs leading to recombination between homologous chromosomes can produce genetic diversity. Endogenous and exogenous sources of DNA lesions resulting in physical impediments to the replication fork can also result in DSBs. Therefore, HR plays a prominent role in the replicative S phase of the mammalian cell cycle, and HR deficiencies can lead to genomic instability [3–5]. The mechanistic steps of HR-dependent repair involve DNA end resection, strand invasion, homology search, and pairing followed by new DNA synthesis.

DNA DSB processing by the HR machinery in mammalian cells is complex, involves several proteins, and RAD51 requires assistance by mediator proteins (e.g., BRCA2) to successfully form the nucleoprotein filament. DNA end resection following a DSB is accomplished by a group of helicases and nucleases including MRE11, BLM/EXOI, and DNA2 among others [6–11]. The resection process ensures commitment to a template-driven HR process rather than ligation of the broken DNA molecules by nonhomologous end-joining (NHEJ) or an alternative end-joining/ssDNA annealing pathway. Immediately following or concurrent with resection, replication protein A (RPA) binds and stabilizes 3′ single-stranded DNA (ssDNA) tails preventing inappropriate annealing or the formation of secondary structures in the DNA. RPA possesses high affinity for ssDNA and can effectively block the next step in HR by competing with RAD51 for binding to the ssDNA substrate. As a mediator protein, BRCA2 can overcome RPA inhibition by delivering RAD51 to the RPA-coated resected ssDNA, displace the RPA, and stabilize growth of the RAD51 filament [7, 12]. RAD51 nucleation, growth, and filament stabilization are followed by the homology search, joint molecule formation, and eventual exchange between the invading ssDNA and the double-stranded DNA (dsDNA) donor template [13].

In this chapter, we describe an in vitro DNA strand exchange reaction with the human RAD51 protein that catalyzes invasion, pairing, and exchange of DNA strands during HR [14–18]. Upon incubation with ssDNA in vitro, purified human RAD51 binds 3 nucleotides per monomer forming a right-handed helical nucleoprotein filament, and remarkably, extends ssDNA 1.5-fold relative to B-form DNA [1]. We outline how RAD51-dependent DNA strand exchange can be stimulated by the mediator protein, BRCA2. BRCA2 by itself is incapable of recombining DNA molecules but can simultaneously load and direct RAD51 to ssDNA whilst inhibiting binding of RAD51 to dsDNA [7]. Furthermore, BRCA2 stabilizes the ensuing RAD51-ssDNA nucleoprotein filament by downregulating the intrinsic ATPase activity of RAD51, preventing turnover and effectively maintaining RAD51 in an ATP-bound form complexed with ssDNA. [19, 20].

2. Materials

2.1. Preparation of DNA

  1. Oligos RJ-167, RJ-PHIX-42–1, RJ-Oligo1, and RJ-Oligo2 were ordered PAGE-purified from Integrated DNA Technology (IDT) [7]. A complete list of oligonucleotides used in this study is provided in Table 1.

  2. 1 M Tris (pH 7.5 and pH 8.0), 1 M Tris acetate (pH 7.5), 0.5 M EDTA (pH 8.0), 5 M NaCl, 1 M MgCl2, 1 M CaCl2, 1 mg/mL bovine serum albumin (BSA), 1 M dithiothreitol (DTT) and 10% SDS stock solutions (see Note 1).

  3. TE buffer: 10 mM Tris (pH 8.0) and 0.1 mM EDTA.

  4. Radioactive ATP[γ−32P] with specific activity of 6000 Ci/mmol (Perkin Elmer).

  5. T4 polynucleotide kinase (PNK) and 10× T4 PNK reaction buffer (New England Biolabs).

  6. Nuclease-free water.

  7. MicroSpin™ G-25 columns (GE Healthcare).

  8. 37 °C and 95 °C dry baths.

  9. Benchtop centrifuge (Eppendorf 5424) and a table topmicrocentrifuge.

  10. Nanodrop® spectrophotometer or other instrument that accurately measures A260 for small quantities of DNA molecules in solution.

Table 1.

Oligonucleotide substrates used for the strand exchange assay

Oligonucleotides Sequence (5′–3′)
RJ-167 CTG CTT TAT CAA GAT AAT TTT TCG ACT CAT CAG AAA TAT CCG TTT CCT ATA TTT ATT CCT ATT ATG TTT TAT TCA TTT ACT TAT TCT TTA TGT TCA TTT TTT ATA TCC TTT ACT TTA TTT TCT CTG TTT ATT CAT TTA CTT ATT TTG TAT TAT CCT TAT CTT ATT TA
RJ-PHIX-42–1 GAC GAA ATA GTT CTA TTA AAA AGC TGA GTA GTC TTT ATA GGC
RJ-Oligo1 (40mer) TAA TAC AAA ATA AGT AAA TGA ATA AAC AGA GAA AAT AAA G
RJ-Oligo2 (40mer) CTT TAT TTT CTC TGT TTA TTC ATT TAC TTA TTT TGT ATT A
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For the DNA strand exchange assay, RJ-167 is annealed to RJ-PHIX-42–1 to make the 3′ tail, and RJ-Oligo1 is labeled with ATP[γ−32P] and then annealed to RJ-Oligo2 to make the donor duplex

2.2. Radiolabeling and Annealing of DNA Strands

  1. 1× T4 PNK reaction buffer from 10× stock.

  2. Annealing buffer (STE buffer): 10 mM Tris–Cl (pH 7.5),100 mM NaCl, and 1 mM EDTA. This buffer can be filtered using a 0.22 μm pore-size filter and stored at room temperature.

  3. 1.5 mL polypropylene microcentrifuge tubes with safety locks.

2.3. DNA Strand Exchange Assay

  1. 1. Adenosine 5′-triphosphate (ATP) (GE Healthcare), pH adjusted to 7.5 (see Note 2).

  2. Assay buffer: 25 mM Tris acetate (pH 7.5), 1 mM MgCl2, 2 mM CaCl2, 0.1 μg/mL BSA, 2 mM ATP and 1 mM DTT. All DNA/protein incubations are performed at 37 C unless otherwise noted (see Notes 3 and 4).

  3. RPA and RAD51 proteins purified from E. coli and BRCA2 purified from human embryonic kidney (HEK) 293T cells [7, 14, 21–23] (see Notes 5 and 6).

  4. RAD51 storage buffer: 50 mM Tris Acetate (pH 7.5), 200 mMKCl, 0.1 mM EDTA, 1 mM DTT, and 50% glycerol. Store at −20 °C (see Note 7).

  5. RPA storage buffer: 20 mM Tris (pH 7.5), 100 mM NaCl,1 mM DTT, 1 mM EDTA, 20% Glycerol. Store at −20 °C.

  6. DNA loading dye (6× concentrate): 10 mM Tris (pH 7.5), 30% glycerol, 0.3% bromophenol blue, and 0.3% xylene cyanol.

  7. Proteinase K, 20 mg/mL stock concentration with a specific activity of ~2 units/mg (Roche) in 0.5% SDS solution.

  8. Make a concentrated (50×) stock solution of TAE by weighing out 242 g of Tris base (FW = 121.14 g/mol) and dissolving it in approximately 750 mL of deionized water. Carefully add 57.1 mL of glacial acid and 100 mL of 0.5 M EDTA (pH 8.0). After that, adjust the solution to a final volume of 1 L. This stock solution can be stored at room temperature. The pH of this buffer is not adjusted and should be about 8.5. To make 1 L of 1 × TAE running buffer: add 20 mL of the 50× TAE stock solution to 980 mL of de-ionized water. This solution can also be stored at room temperature.

  9. 10% ammonium persulfate (APS) solution freshly prepared and tetramethylethylenediamine (TEMED).

  10. 30% acrylamide solution.

  11. 6% polyacrylamide TAE gels: To make a set of five 0.75 mm gels for use with the Hoefer mini vertical gel casting system mix 8 mL of 5× TAE, 8 mL of 30% acrylamide solution, 24 mL of water, 0.24 mL 10% APS, and 0.08 mL of TEMED (see Notes 8 and 9).

  12. BioDot gel loading tips (Dot Scientific Inc).

  13. Hoefer mini vertical gel electrophoresis unit and a powersupply.

  14. DE81 ion exchange chromatography paper (Whatman®).

  15. Blotting paper (VWR).

  16. Polyvinyl wrapping film (Fisher Scientific).

  17. Gel dryer.

  18. PhosphorImager screens and cassettes (GE HealthCare).

  19. PhosphorImager System (Storm 860 or equivalent model,Molecular Dynamics).

  20. ImageQuant software (version 5.1, GE Healthcare).

3. Methods

The DNA strand exchange assay described here recapitulates three key steps in mammalian HR utilizing oligonucleotide length DNA substrates: (1) strand invasion by a RAD51-coated 3′ tail oligonucleotide substrate (RJ-167 annealed to RJ-PHIX-42–1) into a 40b.-p. duplex donor DNA (RJ-Oligo1 annealed to RJ-Oligo2), (2) homologous pairing between the two joint molecules, and (3) exchange resulting in strand pairing between the incoming 3′ tail DNA (RJ-167 annealed to RJ-PHIX-42–1) and the radiolabeled complementary oligonucleotide (RJ-Oligo1) concurrent with displacement of the unlabeled oligonucleotide (RJ-Oligo2). Conceptually, RAD51 facilitates strand exchange between the unlabeled 3′ tail oligonucleotide substrate (consisting of 42 b.p. of dsDNA and a 125-nucleotide ssDNA 3′ tail) and the short 40 b.p. duplex labeled with ATP[γ−32P] as depicted in Fig. 1b. The reaction products appear as a distinct species with higher mobility compared to the short 40 b.p. donor duplex in a 6% nondenaturing polyacrylamide gel (Fig. 1c).

Fig. 1.

Fig. 1

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DNA strand exchange mediated by RAD51 protein. (a) Flowchart of DNA strand exchange assay. (b) Schematic depicting DNA strand exchange protocol. (c) Autoradiogram of DNA strand exchange reactions with the 3′ tail DNA substrate at increasing concentrations of RAD51 (lanes 1–5). (d) Quantification of the data in (a) indicating that optimal exchange occurs at around 0.22 μM RAD51. Error bars represent the S.D. (Reproduced from ref. 7 with permission from Nature Publishing Group)

3.1. Radiolabeling Oligonucleotide RJ-Oligo1

  1. Add appropriate amount of 10 mM Tris–Cl (pH 8.5) buffer to the lyophilized PAGE purified oligonucleotides to make a 10 μM stock which can be stored at −20 °C.

  2. Use A260 reading for the oligonucleotides to determine the correct concentration of the DNA strands using the molar extinction coefficient (ε, M−1 cm−1).

  3. Radiolabel 1 μM of the DNA oligonucleotide (RJ-Oligo1) with ATP[γ−32P] in a 25 μL reaction: add 14 μL of nuclease-free water, 2.5 μL of the 10 μM oligonucleotide, 2.5 μL of 10× PNK buffer, 5 μL of ATP[γ−32P] (~50 μCi), and 1 μL of T4 PNK enzyme, in this order.

  4. Incubate at 37 °C for 60 min.

  5. Heat inactivate the T4 PNK enzyme at 75 °C for 5 min.

  6. During the 5-min heat inactivation step with T4 PNK enzyme, prepare the MicroSpin™ G-25 column.

  7. Resuspend the resin in the column by vortexing.

  8. Loosen the cap one-quarter turn and twist off the bottom closure.

  9. Place the column in the supplied collection tube and spin at 735 × g for 1 min.

  10. Place the column into a fresh DNAse-free 1.5 mL microcentrifuge tube.

  11. Apply the radiolabeled DNA to the MicroSpin™ G-25 column by carefully pipetting the sample to the middle of the packed resin trying not to disrupt or touch the resin with your pipet tip.

  12. Spin the column at 735 × g and retain the eluate, which is the labeled oligo, and store at 4 °C for not more than 14 days for 32 P (see Note 10).

3.2. DNA Strand Annealing to Prepare the 3′ Tail Substrate and the Radiolabeled dsDNA Donor

  1. Add equimolar amounts of RJ-167 and RJ-PHIX-42–1 in annealing buffer to make 100 nM of the 3′ tail DNA.

  2. Add equimolar amounts of radiolabeled RJ-Oligo1 and unlabeled RJ-Oligo2 in annealing buffer to make 100 nM of the radiolabeled duplex donor DNA.

  3. Mix the reactants by gently tapping the tube and spin them briefly at 735 × g for 10–15 s.

  4. Incubate the tubes in a dry bath at 95 °C for 5 min exactly.

  5. After 5 min, turn off the dry bath and allow the annealing reaction to slowly cool to room temperature overnight.

  6. To evaluate the quality of the radiolabeled oligonucleotide substrates and the annealing products, run a small amount on a 6% PAGE gel and follow steps11 through 18 as discussed in Subheading 3.3 (see Note 11).

3.3. Optimizing the RAD51 Concentration in the DNA Strand Exchange Assay

  1. Prepare a reaction cocktail with sufficient volume for the number of samples to be analyzed including the final concentrations of the following: 25 mM Tris-Acetate (pH = 7.5), 1 mM MgCl2, 2 mM CaCl2, 0.1 μg/μL BSA, 2 mM ATP, and 1 mM DTT. These reactions can be assembled at room temperature (~25 °C); however, working stocks of proteins should be kept at 4 °C.

  2. Next, add unlabeled 3′ tail DNA (RJ-167 annealed with RJ-PHIX-42–1) to the cocktail at a final concentration of 4 nM (molecules). Mix each tube gently and then aliquot this reaction mixture into each sample tube for protein addition.

  3. Perform a RAD51 titration to determine the optimal protein: DNA ratio. Add RAD51 protein storage buffer to each reaction in an appropriate volume to compensate for buffer composition (for example, ionic concentrations) across all samples.

  4. Add increasing amounts of RAD51 protein (e.g., 0.1–0.6 μM) for a total volume of 9.6 μL and mix the reactants by gently tapping the tubes.

  5. Incubate the reactions at 37 °C for 5 min.

  6. Remove the reactions from the 37 °C heat block and add 4 nM molecules (e.g., 0.4 μL) of ATP[γ−32P] labeled duplex donor DNA (annealed RJ-Oligo1 and RJ-Oligo2).

  7. Incubate the reaction at 37 °C for 30 min.

  8. During the 30-min incubation with donor dsDNA, wash outthe wells of the 6% PAGE gel extensively with 1× TAE running buffer and pre-run the gel at 60 V.

  9. To terminate the reaction by deproteinization, add 0.5 μL of 0.5% SDS:proteinase K (20 mg/mL) (1:1). Mix the reactants by gently tapping the tube.

  10. Incubate the reaction at 37 °C for 10 min.

  11. Remove the samples from the dry bath and add 1 μL of 6× DNA loading dye to each.

  12. Load the samples into the wells of the 6% polyacrylamide gel using the BioDot gel loading tips and run at 60 V for 70–80 min.

  13. Monitor the migration of the bromophenol blue and xylene cyanol dyes as they progress through the gel, and halt the electrophoresis once the bromophenol blue is approximately 1 cm from the bottom of the gel.

  14. Once the gel has finished running, carefully pour the running buffer down a sink approved for radioactivity disposal.

  15. Disassemble the glass gel plate from the aluminum plate by allowing the gel to remain attached to the aluminum plate (the plastic spacers can be used at this point to gently lift off the glass plate). Place the DE81 paper (cut to the same dimensions as the gel) on top of the gel attached to the aluminum plate, press gently to allow the paper to come in full contact with the gel, and quickly peel the gel away from the aluminum plate retaining the gel now bound to the DE81 paper.

  16. Place the gel face up onto a piece of Whatman® blotting paper cut slightly larger than the DE81 paper (see Fig. 2b), and place a sheet of plastic saran wrap over the gel being careful to remove any wrinkles and place into a vacuum gel dryer for 30 min at 70–80 °C (see Note 12).

  17. After drying, release the vacuum slowly and transfer the driedgel into a cassette and place the phosphorimager screen face down on the gel to expose (lab tape can be used to secure the saran wrap around the Whatman® paper or to affix the gel inside the phosphorimager cassette). The time of exposure can vary from 1 to 24 h depending on the half-life of the 32P isotope (see Note 13).

  18. A phosphorimager system such as the Molecular DynamicsStorm 860 (or equivalent system) can be used to scan the gel (see Note 14).

  19. Finally, analyze and quantify the percentage of the DNA strandexchange product formed by dividing the product over the total input DNA in each lane using ImageQuant software (Fig. 1c, d).

  20. For control experiments, refer to Notes 15–17.

Fig. 2.

Fig. 2

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BRCA2 stimulates DNA strand exchange facilitated by RAD51. (a) Flowchart of DNA strand exchange assay in presence of RPA first followed by BRCA2 and RAD51 proteins. (b) Schematic depicting assembly of the 6% polyacrylamide gel for drying. (c) Autoradiogram of DNA strand exchange reactions in the absence (lanes 1–2) or presence of RPA first (lanes 3–8). Lanes 3–8 depict increasing concentrations of BRCA2 protein (0–40 nM). RAD51 protein concentration is held constant at 0.22 μM. (d) Quantification of the data in (c) Error bars represent the S.D

3.4. RAD51-Mediated DNA Strand Exchange in the Presence of RPA First

  1. Assemble the cocktail reaction as described above in Subheading 3.3 step 1, and aliquot after addition of the 3′ tail DNA to the mix. Then add increasing amounts of RPA protein to a final concentration of 0.02–0.1 μM using storage buffer to compensate for buffer constituents.

  2. Incubate the reaction at 37 °C for 5 min to allow RPA to bind the 3′ tail DNA.

  3. Add RAD51 protein at an optimal concentration predetermined by the RAD51 protein titration experiment in Subheading 3.3.

  4. Incubate the reaction at 37 °C for 5 min.

  5. Continue with steps 6 through 19 as described above in Subheading 3.3.

3.5. RAD51-Mediated DNA Strand Exchange Assay in the Presence of RPA and BRCA2

  1. Add unlabeled 3′ tail DNA (annealed RJ-167 and RJ-PHIX-42–1) to a final concentration of 4 nM (molecules) in assay buffer consisting of 0.1 μM RPA.

  2. Incubate the reaction at 37 °C for 5 min.

  3. Add increasing amounts of BRCA2 protein into each reactionin a range from 2 to 40 nM (see Note 18).

  4. Then add RAD51 protein at the predetermined optimal concentration for activity (e.g., 0.22 μM) for a total volume of 9.6 μL.

  5. Incubate the reaction at 37 °C for 5 min.

  6. Continue with steps 6 through 19 as described above in Subheading 3.3.

  7. For control experiments, refer to Note 18.

Fig. 3.

Fig. 3

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Optimization and control experiments for DNA strand exchange reactions. (a) Autoradiogram showing DNA strand exchange reactions using the 3′ tail DNA substrate in the presence of 0.22 μM RAD51 (lanes 2–7). Standard DNA strand exchange buffer, which contains 2 mM CaCl2, was used. Lane 1: No protein control. Lane 2: ATP omitted. Lane 3: Mg2+ omitted. Lanes 4–7: increasing amounts of RPA were incubated with the DNA substrate for 5 min at 37 °C prior to the addition of RAD51. (b) Quantification of the data in (a). (c) In all DNA strand reactions shown: RPA is 0.1 μM, RAD51 is 0.22 μM, and BRCA2 is 40 nM. Lane 1: no protein control. Lane 2: RPA alone control. Lane 3: BRCA2 alone control. Lane 4: RAD51 alone. Lane 5: BRCA2 and RAD51. Lane 6: RPA and BRCA2. Lane 7: RPA first, RAD51 second. Lane 8: RPA first, BRCA2 and RAD51 second. Lane 9: RPA first, BRCA2 and RAD51 second with tenfold excess cold oligonucleotide complementary to the labeled pairing strand in the donor dsDNA present in the deproteinization step. Lane 10: RPA first, BRCA2 and RAD51 second using a heterologous labeled donor dsDNA. Lane 11: RPA first, BRCA2 and RAD51 second. Lane 12: Same reaction as in lane 11 with ATP omitted. (d) Quantification of the data in (c). (Reproduced from ref. 7 with permission from Nature Publishing Group)

Acknowledgments

This work was supported by grants from the NIH (CA215990-01), American Cancer Society (RSG-17-038-01), the V Foundation BRCA1,2-Convergence Team Award, and a Basser Innovation Award from the Basser Center for BRCA at the University of Pennsylvania Abramson Cancer Center to RBJ. We thank all Jensen lab members for their comments in the preparation of this manuscript.

4 Notes

1.

Use fresh DTT in the reaction buffers. DTT readily oxidizesand absorbs at 280 nm which interferes with spectrophotometric determination of protein concentration [24]. Beta-mercaptoethanol (BME) or Tris(2-carboxyethyl) phosphine hydrochloride (TCEP) solution can be used as a reducing agent in place of DTT.

2.

100 mM aqueous solution of ATP (adenosine 5′-triphosphate), titrated to pH 7.3–7.5 with NaOH. This is stable for 2 years at −20 °C.

3.

Divalent cations such as Ca2+ greatly stimulate the DNA strand exchange activity of human RAD51 protein by reducing the ATP hydrolysis rate, thus maintaining the active RAD51-ATP-ssDNA filament-bound conformation [7, 19, 25]. Therefore, the presence of free Ca2+ ion is essential for this assay.

4.

The presence of Mg2+ is important for RecA and RAD51 orthologues in most reactions that involve protein-DNA interactions [26]. Generally, 1–10 mM MgCl2 or MgSO4 is appropriate for biochemical assays. 1 mM MgCl2 is optimal for human RAD51 in the strand exchange assay reactions described here.

5.

All purified proteins should be aliquoted and stored at −80 °C with appropriate amount of cryoprotectant (e.g., up to 50% glycerol) in the storage buffer.

6.

To prevent loss of activity of the proteins, thaw and dilute aliquots immediately before use and avoid frequent freeze/thaw cycles.

7.

Alternatively, the following recipe can be used as RAD51 storage buffer: 20 mM Hepes (pH = 7.5), 150 mM NaCl, 0.1 mM EDTA, 2 mM BME, and 10% glycerol. Store at −20 °C.

8.

It is advised to make fresh 10% APS solution and 30% acrylamide solution (37.5:1 Acryl:Bis) when casting 6% polyacrylamide gels. The 30% acrylamide solution can be stored at 4 °C for 1–2 months in the dark.

9.

Degas this solution by vacuum for 30 s to remove bubbles.

10.

PAGE-purified oligonucleotides should be used for all stepsthat involve radioactive labeling. Appropriate radiation safety measures should be taken to minimize exposure to radioactivity, and all radioactive waste should be discarded in dedicated solid or liquid waste containers for 32P.

11.

Before performing the DNA strand exchange assay, it is essential to confirm that all DNA substrates have been annealed properly at the correct stoichiometric ratios as unlabeled excess free DNA can impact the final results and complicate data interpretation. Unlabeled substrates (e.g., 3′ tail DNA) can be visualized on a 6% PAGE gel stained with SYBR-Gold while radiolabeled annealed substrates (e.g., 40 b.p. donor dsDNA) can be visualized on a 6% PAGE gel following phosphorimager analysis.

12.

Release the gel dryer vacuum slowly to avoid cracking the gel.Do not over dry the gel as band diffusion can occur.

13.

A Geiger counter can be used to gauge the length of exposure time needed for the gel. If in doubt, scan the gel after 2 h and if the signal is not sufficient, erase the screen and expose the gel for a longer time (12–24 h).

14.

If possible, select “flip” the gel in the horizontal orientation inthe scanner software (as after drying the gel, it will be in the mirror image when scanned from the screen) such that lanes can be read from left to right.

15.

For DNA strand exchange assays, it is imperative to perform control experiments confirming that product formation is not due to melting of the donor duplex DNA and spontaneous annealing during the deproteinization (termination) step. To address this, one can perform a reaction with tenfold excess unlabeled DNA complementary to the labeled pairing strand in the termination mix to demonstrate that the resulting products remain unchanged [7] (Fig. 3c lane 9).

16.

It is important to verify that the stimulation of RAD51 activityis ATP dependent and product formation is inhibited when a heterologous template is substituted into the assay (Fig. 3a lane 2, Fig. 3c lane 10).

17.

Optimal activity should be obtained at a molar protein:DNA nucleotide ratio of 1:3 representing the 3 nt site size bound by each RAD51 monomer at saturation of the DNA lattice (at 4 nM (molecules) of 3′ tail DNA, the optimal RAD51 concentration is 0.22 μM—see Fig. 1c, d).

18.

A control experiment should be performed to demonstrate that BRCA2 alone is not promoting DNA strand exchange as shown in Fig. 3c lane 3.

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