Extended Data Fig. 11: RAD51AP1 promotes the formation of DR-loops in donor DNA.
From: RNA transcripts stimulate homologous recombination by forming DR-loops
a, Experimental design for in vitro DR-loop formation using biotinylated ssRNA and fluorescently labelled ssDNA. b, In vitro DR-loop formation with biotinylated ssRNA (63 nt, 30 nM), RAD51AP1 (1 μM), RAD51 (0.25 μM), fluorescently labelled ssDNA (60 nt, 20 nM) and a dsDNA plasmid. The presence or absence of the indicated reaction components is shown above the dot blot. The levels of ssDNA captured by biotinylated ssRNA via DR-loops were measured by dot blot and quantified over blot background. Data are mean (n = 2–3 independent experiments). c, In vitro DR-loop formation with biotinylated ssRNA (50 nt, 30 nM), RAD51AP1WT (0.5 μM) or RAD51AP1DBM (0.5 μM), RAD51 (0.25 μM), fluorescently labelled ssDNA (50 nt, 20 nM) and a dsDNA plasmid. The presence or absence of the indicated reaction components is shown above the dot blot. The levels of ssDNA captured by biotinylated ssRNA via DR-loops were measured by dot blot and quantified over blot background. Representative results from two similar experiments are shown. d, In vitro DR-loop confirmation with RNaseH treatment. Data are mean (n = 2–3 independent experiments). e, In vitro DR-loop formation with different ssRNA and ssDNA oligos and dsDNA plasmids. Top, schematic of the ssRNA and ssDNA oligos tested. Red, biotinylated ssRNA; purple, fluorescently labelled ssDNA. The lengths and relative annealing positions of the oligos are indicated. pBSK+ and pMLM-mini are two different dsDNA plasmids that were used in the reactions. Both of them contain sequences homologous to the ssRNA and ssDNA oligos. f, Schematic of the three ssDNA oligos (red) and the ssRNA oligo (green) used in Fig. 5c, d. g, In vitro D-loop formation with RAD51 and ssDNA oligos A–C. RAD51 (0.25 μM) was incubated with labelled 60-nt ssDNA oligos A, B or C (30 nM) and then with a dsDNA plasmid containing sequences homologous to oligos A and B but not C. Formation of D-loops was analysed by native gel electrophoresis. The efficiency of D-loop formation was determined by quantifying the shifted and unshifted bands in a light exposure of the gel. Data are mean ± s.d. (n = 3 independent experiments).
