Figure 7

KCl induced changes in the aggregation of RAD51-dsDNA complexes: protein and DNA analyses. Relaxed φX174 DNA (30 μM) along with Topo I was incubated with RAD51 (3.0 μM) in presence of increasing concentration of KCl [0 mM, 25 mM, 50 mM, 100 mM, 140 mM, & 200 mM] with 1 mM ATP either in presence of RAD52 [2.0 μM] (Panel B: Protein analysis, Panel D:DNA analysis) or in its absence (Panel A: Protein analysis, Panel C:DNA analysis) in binding buffer R at 37°C for 12 minutes followed by centrifugation assay (Methods) and analyses of proteins by SDS-PAGE (Methods). Lanes s1–s6 and p1–p6 in all the Panels represent supernatant and pellet fractions for increasing concentrations of KCl respectively. RAD51 bands in pellet fraction is expressed as a fraction of the protein band intensities associated with the sum of supernatant and pellet fractions and plotted as a function of KCl concentration [(Open square: Panel E) in presence of RAD52] [(Open diamond: Panel E) in absence of RAD52)]. Similarly RAD52 protein is also represented in Panel E (open triangle). For DNA analysis, samples were deproteinized (Methods). Percentage of DNA (relaxed and unwound forms) in pellet fraction was expressed as a ratio of the total DNA (relaxed and unwound forms) in both pellet and supernatant fractions and plotted as a function of KCl concentration [(Filled square: Panel E) presence of RAD52 & (Filled diamond: Panel E) absence of RAD52].