Fig. 3: SWSAP1-SWS1 stimulates RAD51 D-loop activity on RPA-coated ssDNA.

A Schematic depicting D-loop assay. FITC-90mer ssDNA (yellow DNA with circle) was coated with RPA (green circles) for 5 min at 37 °C. Subsequently, RAD51 (orange circles), SWSAP1-SWS1 (pink and purple circles), or RAD51 with SWSAP1-SWS1 was added to the reaction for 5 min at 37 °C and then incubated with the pBluscript dsDNA template (supercoiled DNA) for 15 min at 37 °C. D-loops were deproteinated, run on an agarose gel, and imaged for FITC signal. B FITC-90mer ssDNA coated with RPA (100 nM) was challenged with either RAD51 (0.25, 0.5, 1 μM) or RAD51 (0.25 μM) and increasing concentrations of SWSAP1-SWS1 as indicated. Then the reaction was begun by adding pBluescript dsDNA. D-loops were deproteinated, run on an agarose gel, and imaged for FITC signal. DNA alone, DNA + RPA, DNA + RAD51, DNA + RAD51 + RPA are controls (Lanes 1-6). C Quantification of D-loop reaction products with recombinant SWSAP1-SWS1. The reactions utilized 100 nM RPA, 250 nM RAD51, and SWSAP1-SWS1 (1.25, 2.5, 62.5, and 125 nM) proteins and the values were normalized to RPA 100 nM and RAD51 250 nM. Experiments were performed five times, and standard deviations are shown.