Figure 1

A) Schematic for DNA unwinding assay. Image modified from Chi et al (Fig. 4A) [17]. RAD51 is incubated with relaxed dsDNA along with Topo I. RAD51 upon binding to dsDNA causes dsDNA unwinding generating compensatory overwinding elsewhere in the same DNA molecule. Topo I acts as a "swivel" releasing excess turns thereby capturing the events of DNA unwinding that can be monitored after deproteinization by agarose gel electrophoresis. (B-I) RAD51 unwinds dsDNA in presence of various nucleotide cofactors. Relaxed φX174DNA (30 μM) along with Topo I was incubated with increasing concentrations of RAD51 (3 μM, 6 μM & 9 μM) (lanes 1–3 in Panels C – H) in binding buffer R [30 mM Tris-HCl (pH 7.6), 1 mM DTT, 1 mM MgCl₂] at 37°C for 12 minutes, in presence of either ATP, ADP, AMP-PNP, ATPγS, or AMP (1 mM each) (Panels C – G) or no nucleotide (Panel H), followed by deproteinization and agarose gel assay (Methods). Panel B represents no protein control. Intensity of DNA bands was quantified using Image J software. Percentage unwound DNA (background-corrected and quantified as explained in Materials and methods) was expressed as a ratio of the intensity associated with unwound DNA band to the sum of intensities corresponding to unwound as well as relaxed dsDNA bands and plotted as a function of RAD51 concentration (Panel I). Error bars denote standard deviation in data values across three independent experiments. No significant variation was observed between experiments (2 tailed t-test, P < 0.05).