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

Abstract

Human Rad51 (hRad51) protein plays a key role in homologous recombination and DNA repair. hRad51 protein forms a helical filament on single-stranded DNA (ssDNA), which performs the basic steps of homologous recombination: a search for homologous double-stranded DNA (dsDNA) and DNA strand exchange. hRad51 protein possesses DNA-dependent ATPase activity; however, the role of this activity has not been understood. Our current results show that Ca(2+) greatly stimulates DNA strand exchange activity of hRad51 protein. We found that Ca(2+) exerts its stimulatory effect by modulating the ATPase activity of hRad51 protein. Our data demonstrate that, in the presence of Mg(2+), the hRad51-ATP-ssDNA filament is quickly converted to an inactive hRad51-ADP-ssDNA form, due to relatively rapid ATP hydrolysis and slow dissociation of ADP. Ca(2+) maintains the active hRad51-ATP-ssDNA filament by reducing the ATP hydrolysis rate. These findings demonstrate a crucial role of the ATPase activity in regulation of DNA strand exchange activity of hRad51 protein. This mechanism of Rad51 protein regulation by modulating its ATPase activity is evolutionarily recent; we found no such mechanism for yeast Rad51 (yRad51) protein.

Fig. 1.

Effect of divalent cations on DNA strand exchange activity of hRad51 protein. (a) The hRad51 nucleoprotein filaments were formed on ssDNA (94-mer, no. 71); DNA strand exchange was initiated by addition of dsDNA (32-mer, nos. 5 and 6). Asterisks denote the ³²P label. The products of DNA strand exchange resolved by gel-electrophoresis are shown at the Bottom. The cation concentrations were 5 mM. (b) The effect of cation concentrations on the extent of DNA strand exchange. (Inset) The effect of Ca²⁺ (10 mM) and Mg²⁺ (20 mM) on DNA strand exchange promoted by yRad51 protein is shown as a graph.

Fig. 2.

Ca²⁺ stimulates D-loop formation promoted by hRad51 protein. (a) The scheme and the time course of D-loop formation analyzed in a 1% agarose gel. An asterisk denotes the ³²P label. hRad51 protein was preincubated with ssDNA (90-mer, no. 90) either for 10 min at 1 mM MgCl₂ (denoted “Mg²⁺”); for 10 min at 1 mM CaCl₂ (denoted “Ca²⁺”); or for 10 min at 1 mM MgCl₂, and then for 10 min at 1 mM MgCl₂ and 1 mM CaCl₂ (denoted “Mg²⁺ → Mg²⁺ + Ca²⁺”). The reactions in which ATP was omitted are denoted as “-ATP”. The reactions were carried out for the time period indicated by the numbers above the gel. (b) The results of a and the time coarse of D-loop formation promoted by yRad51 protein in the presence of 10 mM Ca²⁺ (open circles) and 20 mM Mg²⁺ (open squares) are shown as a graph.

Fig. 3.

Effect of Ca²⁺ concentrations on the efficiency of D-loop formation. The human and yeast Rad51-ssDNA filaments (filled and open circles, respectively) were formed by incubation of the protein with ³²P-labeled ssDNA (90-mer, no. 90) in the presence of indicated Ca²⁺ concentrations. JM formation was initiated by addition of pUC19 dsDNA and carried out for 10 min.

Fig. 4.

Ca²⁺ stimulates DNA three-strand exchange promoted by hRad51 protein. (a) The scheme and the time course of DNA stand exchange between φX174 circular ssDNA and linear dsDNA promoted by hRad51 protein analyzed in a 1% agarose gel. The reactions were carried out at 1 mM Mg²⁺ (denoted “Mg²⁺”); 1 mM Mg²⁺ and 2 mM Ca²⁺ (denoted “Mg²⁺ + Ca²⁺”); 1 mM Mg²⁺ and 2 mM Ca²⁺, but without ATP (denoted “-ATP”); DNA strand exchange promoted by RecA protein is shown as a control (denoted “RecA”). “M” denotes DNA migration markers. (b) The results are shown as a graph. JM and NC formation in the presence of both Ca²⁺ and Mg²⁺ are indicated by squares and circles, respectively. JM formation in the presence of Mg²⁺ is indicated by open circles.

Fig. 5.

Ca²⁺ increases the stability of the hRad51-ssDNA filament. (a) The fluorescence of hRad51-ATP-εDNA complexes declines in the presence of Mg²⁺, but not Ca²⁺. (b) The fluorescence of yRad51-ATP-εDNA complexes does not show such a decline in the presence of Mg²⁺. (c) hRad51-ATP-εDNA complexes show higher resistance to NaCl in the presence of Ca²⁺ than in the presence of Mg²⁺ whereas (Inset) hRad51-ADP-εDNA complexes show the same resistance in the presence of Ca²⁺ and Mg²⁺.

Fig. 6.

Ca²⁺ averts conversion of the hRad51-ATP-ssDNA filament into a hRad51-ADP-ssDNA form by inhibiting ATP hydrolysis. (a) The scheme and results of analysis by filter-binding and TLC of accumulation of the hRad51-ADP-ssDNA complexes in the presence of Mg²⁺ (lanes 1–8); Ca²⁺ (lanes 9–14); or Mg²⁺ followed by addition of Ca²⁺ (lanes 15–22). The content of ATP and ADP in the reaction mixtures (without filter binding) containing either Mg²⁺ or Ca²⁺ is shown in lanes 23–25 and 26–28, respectively. (b) Graphical representation of three independent experiments, as that in a.(c) Inhibition by Ca²⁺ of ATP hydrolysis by the hRad51-ssDNA filament.