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. 2004 Jul 6;101(27):9988-93.
doi: 10.1073/pnas.0402105101. Epub 2004 Jun 28.

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

Dmitry V Bugreev  1 , Alexander V Mazin
Affiliations

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

Dmitry V Bugreev et al. Proc Natl Acad Sci U S A. .

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.

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Figures

Fig. 1.
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.
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.
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.
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.
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.
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.
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