Skip to main content
Figure 5 | BMC Biochemistry

Figure 5

From: Human Rad51 mediated DNA unwinding is facilitated by conditions that favour Rad51-dsDNA aggregation

Figure 5

RAD52 promotes coaggregation of RAD52-RAD51-DNA complexes: centrifugation assay followed by protein analyses. Relaxed Ļ†X174 DNA (30 Ī¼M) along with Topo I was incubated with RAD51 (3.0 Ī¼M) in presence of increasing concentration of RAD52 (0 Ī¼M, 0.5 Ī¼M, 1.0 Ī¼M & 2.0 Ī¼M) in binding buffer R at 37Ā°C for 12 minutes, in presence of either ATP, ADP, AMP-PNP, ATPĪ³S, or AMP (1 mM each) (Panels E-I) or no nucleotide (Panel J), followed by centrifugation assay (Methods) and analyses of proteins by SDS-PAGE (Methods). Lanes s1ā€“s4 and p1ā€“p4 in all the Panels represent supernatant and pellet fractions for increasing concentrations of RAD52 respectively. Lanes 1 & 2 of Panel A show input RAD52 and RAD51 respectively used in centrifugation assay. No DNA controls: lanes s1 and p1 of Panel C correspond to supernatant and pellet fractions for RAD51 (3 Ī¼M) and RAD52 (2 Ī¼M) together, while the same for Panels B and D correspond to RAD52 protein alone and RAD51 protein alone, respectively. Intensity of protein bands was quantified using Image J software. Percentage of RAD51 protein in pellet was expressed as a ratio of the total intensity associated with pellet and supernatant protein bands and plotted as a function of RAD52 protein concentration (Panel K) (percentage of protein in supernatant fraction will therefore be 100% minus pellet fraction). The quantitative data has been given for RAD52 corresponding to only ATP set.

Back to article page