Ca²⁺ activates human homologous recombination protein Rad51 by modulating its ATPase activity

Bugreev and Mazin. 10.1073/pnas.0402105101.

Supporting Information

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Supporting Figure 7
Supporting Figure 8
Supporting Figure 9
Supporting Figure 10

Supporting Figure 7

Fig. 7. Effect of Mg²⁺ on Ca²⁺-dependent DNA three-strand exchange promoted by human Rad51 (hRad51) protein. The products of DNA strand exchange between fX174 circular single-stranded DNA (ssDNA) and linear double-stranded DNA (dsDNA) promoted by hRad51 protein were analyzed in a 1% agarose gel. The reactions were carried out at 2 mM Ca²⁺ and 0, 0.1, 0.2, 0.5, 1, 2, 5, 10, and 20 mM of Mg²⁺ for 1 h under standard conditions, except that concentration of NaCl was 50 mM .

Supporting Figure 8

Fig. 8. Ca²⁺ exerts its stimulatory effect during filament assembly. Joint molecules (JM) (D-loops) formation was carried out, essentially, as described in Materials and Methods. hRad51 protein was incubated with ssDNA in the presence of 5 mM Mg²⁺ for 10 min; then, the mixture was divided in two. To the first of them (bar 1), 5 mM Ca²⁺ was added, followed by additional incubation for 10 min. Then, JM formation was initiated by addition of pUC19 dsDNA and carried out for 10 min. In the second mixture (bar 2), JM formation was initiated immediately by addition of pUC19 dsDNA and carried out for 5 min. Then, 5 mM Ca²⁺ was added, and the reaction was continued for another 10 min. The extent of JM formation is shown as a bar graph. The extent of joint molecules in the reaction (bar 1) is expressed as 100%.

Supporting Figure 9

Fig. 9. Effect of the ATP-regeneration system (9 mM phoshocreatine and 10 units/ml phosphocreatine kinase) on accumulation of ADP within the hRad51-ssDNA nucleoprotein in the presence of Mg²⁺. Graphical representation of the kinetics of ADP accumulation either in the absent (filled circles), or in the presence (open triangles) of the ATP regeneration system. The fraction of the protein-bound ADP was determined using the filter-binding assay. In parallel, the activity of the ATP regeneration was tested and found to be sufficient to completely remove all ADP from solution.

Supporting Figure 10

Fig. 10. Nonhydrolyzable ATP-analogs efficiently support DNA strand exchange activity of Rad51 in the presence of either Ca²⁺ or Mg²⁺. The hRad51-ssDNA nucleoprotein filaments were formed by incubation with ssDNA in the presence of either 2 mM Mg²⁺ or 2 mM Ca²⁺ and 2 mM of one of the nucleotide cofactors for 10 min. The following nucleotide cofactors were used: ATP, AMP-PNP, or AMP-PCP. JMs were initiated by addition of pUC19 dsDNA and were carried out for 10 min.