Choosing a strategy for designing of branched DNA substrates for BM
1| The substrates for BM can be prepared by mixing and annealing of two simple DNA intermediates (forked, tailed and ssDNAs) (Fig. 2).
2| When constructing the substrates with four arms (X-junctions, PX-junctions), it is important that one of the arms is non-homologous relative to three others (Fig. 2a, b shaded), otherwise it would be impossible to prepare forked DNA intermediates.
3| To block spontaneous BM6, 7 at least a single base pair heterology has to be introduced into duplexes involved in BM (Fig. 1b). The substrates for 3-stranded BM reaction should have at least four bases of heterology, because shorter regions of heterology are not sufficient to block spontaneous 3-stranded BM, which proceeds more efficiently than spontaneous BM on 4-stranded DNA substrates6, 7. When constructing PX-junctions or replication forks a heterologous base should be placed at least several bases away from the junction.
Preparation of substrates for BM
1| Purify the individual oligonucleotides by electrophoresis in a denaturing polyacrylamide gel. Label one of the oligonucleotides using [γ-32P]ATP and T4 polynucleotide kinase, followed by DNA purification through the Micro-BioSpin 6 column (Bio-Rad). Determine concentrations of all oligonucleotides spectrophotometrically using known molar extinction coefficients (can be obtained from the IDT website www.idtdna.com).
2| To prepare tailed or forked DNA intermediates (Fig. 2), mix equal amounts of each of the complementary ssDNA strands (usually 1 µM of molecules) in 1 x SSC buffer, heat for 3 min at 95°C, and allow annealing to proceed for 1 hour at optimal hybridization temperature (Th). Calculate Th using the formula: Th=1.24 x Tm-43.8; where Tm is a melting temperature of the double-stranded part of the resulting structure. Calculate the Tm using a tool from the Promega website (www.promega.com/biomath/calc11.htm).
Testing of BM activity of proteins
1| Test BM activity in a range of protein concentrations by adding increasing amounts of your protein to the annealed BM structure (e.g. BM activity of Rad54 protein can be detected in 5-1000 nM range) and incubating the mixture for the desirable time at the reaction temperature.
2| Measure the time course of the BM reaction at the appropriate protein concentrations.
Separation of BM products in polyacrylamide gel
1| Stop the reaction by addition of SDS (to 1.5%), place on ice, mix with a 1/10 volume of loading buffer (70% glycerol, 0.1% bromophenol blue) and analyze by electrophoresis in native 5-10% polyacrylamide gels in 1 x TBE. Run the gel at 11 V/cm for 1.5-2 hours. Some proteins form very stable complexes with DNA whose disruption requires treatment with SDS (1.5%) and proteinase K (800 µg/ml) for 1-15 min at room temperature before electrophoresis. Alternatively stop the reaction by addition of 1/10 of the reaction volume of polydT (1 mg/ml, GE Healthcare) followed by 1 min incubation at 23°C and by deproteinization with SDS and proteinase K, as described above. Concentration and acrylamide/bis-acrylamide ratio of polyacrylamide gels may vary depending on the size and structure of BM substrates. Some structures (e.g. X-junctions) become unstable during electrophoresis at room temperature and should be resolved at 4°C.
2| Dry the gel on DE81 chromatography paper, visualize and quantify the products of BM using a PhosphorImager system (GE Healthcare), or any other appropriate device.