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Rad51 and Rad54 ATPase activities are both required to modulate Rad51-dsDNA filament dynamics.

Li X, Zhang XP, Solinger JA, Kiianitsa K, Yu X, Egelman EH, Heyer WD - Nucleic Acids Res. (2007)

Bottom Line: The results with Rad51-K191R mutant protein also revealed an unexpected defect in binding to DNA.Once formed, Rad51-K191R-DNA filaments appeared normal upon electron microscopic inspection, but displayed significantly increased stability.These biochemical defects in the Rad51-K191R protein could lead to deficiencies in presynapsis (filament formation) and postsynapsis (filament disassembly) in vivo.

View Article: PubMed Central - PubMed

Affiliation: Section of Microbiology, University of California, Davis, CA 95616-8665, USA.

ABSTRACT
Rad51 and Rad54 are key proteins that collaborate during homologous recombination. Rad51 forms a presynaptic filament with ATP and ssDNA active in homology search and DNA strand exchange, but the precise role of its ATPase activity is poorly understood. Rad54 is an ATP-dependent dsDNA motor protein that can dissociate Rad51 from dsDNA, the product complex of DNA strand exchange. Kinetic analysis of the budding yeast proteins revealed that the catalytic efficiency of the Rad54 ATPase was stimulated by partial filaments of wild-type and Rad51-K191R mutant protein on dsDNA, unambiguously demonstrating that the Rad54 ATPase activity is stimulated under these conditions. Experiments with Rad51-K191R as well as with wild-type Rad51-dsDNA filaments formed in the presence of ATP, ADP or ATP-gamma-S showed that efficient Rad51 turnover from dsDNA requires both the Rad51 ATPase and the Rad54 ATPase activities. The results with Rad51-K191R mutant protein also revealed an unexpected defect in binding to DNA. Once formed, Rad51-K191R-DNA filaments appeared normal upon electron microscopic inspection, but displayed significantly increased stability. These biochemical defects in the Rad51-K191R protein could lead to deficiencies in presynapsis (filament formation) and postsynapsis (filament disassembly) in vivo.

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Both Rad51 and Rad54 ATPase activities are important for Rad51-dsDNA filament disassembly. Rad51-dsDNA filaments were formed at 4 bp/Rad51 ratio (1.5 μM Rad51, 6 μM dsDNA) for 15 min at 30°C. Initiation of Rad51-dsDNA filament disassembly was started by the addition of Rad54 (0.1 µM) and scavenger DNA (30 µM). (A) Protein-free, labeled DNA substrate (lane 1); Rad51-dsDNA filaments in the absence of Rad54 but with scavenger DNA (lanes 2–5), in the presence of Rad54 and scavenger DNA (lanes 6–9), in the presence of Rad54 but absence of scavenger DNA (lanes 10–13), in the absence of Rad54 and scavenger DNA (lanes 14–17). (B) The data from (A) and from assays where Rad54-K341R was used instead of wild-type Rad54 protein (data not shown) were quantified on a PhosphoImager. The accumulation of the band of free DNA and the reduction of lowest mobility species of the nucleoprotein filament (saturated complex) were taken as the readout of Rad51 displacement activity by Rad54, respectively. Data points represent the average of at least three replicate experiments, and error bars represent 1 SD. For the reactions with Rad51 and Rad51+Rad54 only the 90 min data are shown. (C) Rad51-K191R-dsDNA filaments were formed at 4 bp/Rad51-K191R ratio (1.5 μM Rad51-K191R, 6 μM dsDNA) for 15 min at 30°C. Initiation of Rad51-K191R-dsDNA filament disassembly was started by the addition of Rad54 (0.1 µM) and scavenger DNA (30 µM). Protein-free, labeled DNA substrate (lane 1); Rad51-K191R-dsDNA filaments in the absence of Rad54 but with scavenger DNA (lanes 2–5), in the presence of Rad54 and scavenger DNA (lanes 6–9), in the presence of Rad54 but absence of scavenger DNA (lanes 10–13), in the absence of Rad54 and scavenger DNA (lanes 14–17). (D) The data from (C) were quantified on a PhosphoImager. The reduction of the saturated nucleoprotein filament was taken as readout of Rad51-K191R displacement by Rad54. Data points represent the average of at least three replicate experiments, and error bars represent 1 SD. For the reactions with Rad51-K191R, Rad51-K191R+Rad54 and Rad51-K191R+Rad54-K341R+scavenger DNA only the 90 min data are shown.
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Figure 4: Both Rad51 and Rad54 ATPase activities are important for Rad51-dsDNA filament disassembly. Rad51-dsDNA filaments were formed at 4 bp/Rad51 ratio (1.5 μM Rad51, 6 μM dsDNA) for 15 min at 30°C. Initiation of Rad51-dsDNA filament disassembly was started by the addition of Rad54 (0.1 µM) and scavenger DNA (30 µM). (A) Protein-free, labeled DNA substrate (lane 1); Rad51-dsDNA filaments in the absence of Rad54 but with scavenger DNA (lanes 2–5), in the presence of Rad54 and scavenger DNA (lanes 6–9), in the presence of Rad54 but absence of scavenger DNA (lanes 10–13), in the absence of Rad54 and scavenger DNA (lanes 14–17). (B) The data from (A) and from assays where Rad54-K341R was used instead of wild-type Rad54 protein (data not shown) were quantified on a PhosphoImager. The accumulation of the band of free DNA and the reduction of lowest mobility species of the nucleoprotein filament (saturated complex) were taken as the readout of Rad51 displacement activity by Rad54, respectively. Data points represent the average of at least three replicate experiments, and error bars represent 1 SD. For the reactions with Rad51 and Rad51+Rad54 only the 90 min data are shown. (C) Rad51-K191R-dsDNA filaments were formed at 4 bp/Rad51-K191R ratio (1.5 μM Rad51-K191R, 6 μM dsDNA) for 15 min at 30°C. Initiation of Rad51-K191R-dsDNA filament disassembly was started by the addition of Rad54 (0.1 µM) and scavenger DNA (30 µM). Protein-free, labeled DNA substrate (lane 1); Rad51-K191R-dsDNA filaments in the absence of Rad54 but with scavenger DNA (lanes 2–5), in the presence of Rad54 and scavenger DNA (lanes 6–9), in the presence of Rad54 but absence of scavenger DNA (lanes 10–13), in the absence of Rad54 and scavenger DNA (lanes 14–17). (D) The data from (C) were quantified on a PhosphoImager. The reduction of the saturated nucleoprotein filament was taken as readout of Rad51-K191R displacement by Rad54. Data points represent the average of at least three replicate experiments, and error bars represent 1 SD. For the reactions with Rad51-K191R, Rad51-K191R+Rad54 and Rad51-K191R+Rad54-K341R+scavenger DNA only the 90 min data are shown.

Mentions: Rad54 disassembles Rad51-dsDNA filaments, and we suggested this function to be critical during postsynapsis to turnover the Rad51-heteroduplex DNA product complex (30,41). This disassembly was shown to require the Rad54 ATPase activity (41), but the requirement for the Rad51 ATPase remained unaddressed. To test a possible contribution of the Rad51 ATPase activity, we tested disassembly of Rad51-K191R-dsDNA filaments. As shown before, the Rad51-DNA filament disassembled gradually (Figure 4A, lanes 2–5). A slight accumulation of free dsDNA and reduction of the saturated filament was observed after 90 min of incubation (Figure 4A and B). Visualizing disassembly was dependent on the presence of scavenger DNA (lanes 14–17), showing that disassociated Rad51 can rebind the dsDNA. Addition of Rad54 at a sub-stoichiometric ratio (1 Rad54 per 15 Rad51) promoted nucleoprotein filament disassembly (Figure 4A, lanes 6–9). A significant accumulation of free dsDNA and reduction of the saturated filament was observed after 90 min of incubation (Figure 4A and B). Again, the presence of scavenger DNA was required, since Rad51 can rebind to dsDNA even in the presence of Rad54 (lanes 10–13). In contrast, Rad51-K191R-dsDNA filaments were extremely stable even in the presence of scavenger DNA (Figure 4C, lanes 2–5, Figure 4D), consistent with the results from the salt titrations (Figure 2D). Upon addition of Rad54, the Rad51-K191R-dsDNA filament was partially disassembled, and a gradual increase in the mobility of the nucleoprotein filament was observed during the time course (Figure 4C, lanes 6–9, Figure 4D). The presence of scavenger DNA was required to prevent Rad51-K191R to rebind the substrate (Figure 4C, lane 10–17). Furthermore, Rad54-K341R, an ATPase-deficient mutant, has no effect on either Rad51- or Rad51-K191R-dsDNA filament disassembly [(41), Figure 4D]. Together, these results suggest that the intrinsic ATPase activity of Rad51 and the ATPase activity of Rad54 are both required for the efficient turnover of Rad51 from dsDNA substrate.Figure 4.


Rad51 and Rad54 ATPase activities are both required to modulate Rad51-dsDNA filament dynamics.

Li X, Zhang XP, Solinger JA, Kiianitsa K, Yu X, Egelman EH, Heyer WD - Nucleic Acids Res. (2007)

Both Rad51 and Rad54 ATPase activities are important for Rad51-dsDNA filament disassembly. Rad51-dsDNA filaments were formed at 4 bp/Rad51 ratio (1.5 μM Rad51, 6 μM dsDNA) for 15 min at 30°C. Initiation of Rad51-dsDNA filament disassembly was started by the addition of Rad54 (0.1 µM) and scavenger DNA (30 µM). (A) Protein-free, labeled DNA substrate (lane 1); Rad51-dsDNA filaments in the absence of Rad54 but with scavenger DNA (lanes 2–5), in the presence of Rad54 and scavenger DNA (lanes 6–9), in the presence of Rad54 but absence of scavenger DNA (lanes 10–13), in the absence of Rad54 and scavenger DNA (lanes 14–17). (B) The data from (A) and from assays where Rad54-K341R was used instead of wild-type Rad54 protein (data not shown) were quantified on a PhosphoImager. The accumulation of the band of free DNA and the reduction of lowest mobility species of the nucleoprotein filament (saturated complex) were taken as the readout of Rad51 displacement activity by Rad54, respectively. Data points represent the average of at least three replicate experiments, and error bars represent 1 SD. For the reactions with Rad51 and Rad51+Rad54 only the 90 min data are shown. (C) Rad51-K191R-dsDNA filaments were formed at 4 bp/Rad51-K191R ratio (1.5 μM Rad51-K191R, 6 μM dsDNA) for 15 min at 30°C. Initiation of Rad51-K191R-dsDNA filament disassembly was started by the addition of Rad54 (0.1 µM) and scavenger DNA (30 µM). Protein-free, labeled DNA substrate (lane 1); Rad51-K191R-dsDNA filaments in the absence of Rad54 but with scavenger DNA (lanes 2–5), in the presence of Rad54 and scavenger DNA (lanes 6–9), in the presence of Rad54 but absence of scavenger DNA (lanes 10–13), in the absence of Rad54 and scavenger DNA (lanes 14–17). (D) The data from (C) were quantified on a PhosphoImager. The reduction of the saturated nucleoprotein filament was taken as readout of Rad51-K191R displacement by Rad54. Data points represent the average of at least three replicate experiments, and error bars represent 1 SD. For the reactions with Rad51-K191R, Rad51-K191R+Rad54 and Rad51-K191R+Rad54-K341R+scavenger DNA only the 90 min data are shown.
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Figure 4: Both Rad51 and Rad54 ATPase activities are important for Rad51-dsDNA filament disassembly. Rad51-dsDNA filaments were formed at 4 bp/Rad51 ratio (1.5 μM Rad51, 6 μM dsDNA) for 15 min at 30°C. Initiation of Rad51-dsDNA filament disassembly was started by the addition of Rad54 (0.1 µM) and scavenger DNA (30 µM). (A) Protein-free, labeled DNA substrate (lane 1); Rad51-dsDNA filaments in the absence of Rad54 but with scavenger DNA (lanes 2–5), in the presence of Rad54 and scavenger DNA (lanes 6–9), in the presence of Rad54 but absence of scavenger DNA (lanes 10–13), in the absence of Rad54 and scavenger DNA (lanes 14–17). (B) The data from (A) and from assays where Rad54-K341R was used instead of wild-type Rad54 protein (data not shown) were quantified on a PhosphoImager. The accumulation of the band of free DNA and the reduction of lowest mobility species of the nucleoprotein filament (saturated complex) were taken as the readout of Rad51 displacement activity by Rad54, respectively. Data points represent the average of at least three replicate experiments, and error bars represent 1 SD. For the reactions with Rad51 and Rad51+Rad54 only the 90 min data are shown. (C) Rad51-K191R-dsDNA filaments were formed at 4 bp/Rad51-K191R ratio (1.5 μM Rad51-K191R, 6 μM dsDNA) for 15 min at 30°C. Initiation of Rad51-K191R-dsDNA filament disassembly was started by the addition of Rad54 (0.1 µM) and scavenger DNA (30 µM). Protein-free, labeled DNA substrate (lane 1); Rad51-K191R-dsDNA filaments in the absence of Rad54 but with scavenger DNA (lanes 2–5), in the presence of Rad54 and scavenger DNA (lanes 6–9), in the presence of Rad54 but absence of scavenger DNA (lanes 10–13), in the absence of Rad54 and scavenger DNA (lanes 14–17). (D) The data from (C) were quantified on a PhosphoImager. The reduction of the saturated nucleoprotein filament was taken as readout of Rad51-K191R displacement by Rad54. Data points represent the average of at least three replicate experiments, and error bars represent 1 SD. For the reactions with Rad51-K191R, Rad51-K191R+Rad54 and Rad51-K191R+Rad54-K341R+scavenger DNA only the 90 min data are shown.
Mentions: Rad54 disassembles Rad51-dsDNA filaments, and we suggested this function to be critical during postsynapsis to turnover the Rad51-heteroduplex DNA product complex (30,41). This disassembly was shown to require the Rad54 ATPase activity (41), but the requirement for the Rad51 ATPase remained unaddressed. To test a possible contribution of the Rad51 ATPase activity, we tested disassembly of Rad51-K191R-dsDNA filaments. As shown before, the Rad51-DNA filament disassembled gradually (Figure 4A, lanes 2–5). A slight accumulation of free dsDNA and reduction of the saturated filament was observed after 90 min of incubation (Figure 4A and B). Visualizing disassembly was dependent on the presence of scavenger DNA (lanes 14–17), showing that disassociated Rad51 can rebind the dsDNA. Addition of Rad54 at a sub-stoichiometric ratio (1 Rad54 per 15 Rad51) promoted nucleoprotein filament disassembly (Figure 4A, lanes 6–9). A significant accumulation of free dsDNA and reduction of the saturated filament was observed after 90 min of incubation (Figure 4A and B). Again, the presence of scavenger DNA was required, since Rad51 can rebind to dsDNA even in the presence of Rad54 (lanes 10–13). In contrast, Rad51-K191R-dsDNA filaments were extremely stable even in the presence of scavenger DNA (Figure 4C, lanes 2–5, Figure 4D), consistent with the results from the salt titrations (Figure 2D). Upon addition of Rad54, the Rad51-K191R-dsDNA filament was partially disassembled, and a gradual increase in the mobility of the nucleoprotein filament was observed during the time course (Figure 4C, lanes 6–9, Figure 4D). The presence of scavenger DNA was required to prevent Rad51-K191R to rebind the substrate (Figure 4C, lane 10–17). Furthermore, Rad54-K341R, an ATPase-deficient mutant, has no effect on either Rad51- or Rad51-K191R-dsDNA filament disassembly [(41), Figure 4D]. Together, these results suggest that the intrinsic ATPase activity of Rad51 and the ATPase activity of Rad54 are both required for the efficient turnover of Rad51 from dsDNA substrate.Figure 4.

Bottom Line: The results with Rad51-K191R mutant protein also revealed an unexpected defect in binding to DNA.Once formed, Rad51-K191R-DNA filaments appeared normal upon electron microscopic inspection, but displayed significantly increased stability.These biochemical defects in the Rad51-K191R protein could lead to deficiencies in presynapsis (filament formation) and postsynapsis (filament disassembly) in vivo.

View Article: PubMed Central - PubMed

Affiliation: Section of Microbiology, University of California, Davis, CA 95616-8665, USA.

ABSTRACT
Rad51 and Rad54 are key proteins that collaborate during homologous recombination. Rad51 forms a presynaptic filament with ATP and ssDNA active in homology search and DNA strand exchange, but the precise role of its ATPase activity is poorly understood. Rad54 is an ATP-dependent dsDNA motor protein that can dissociate Rad51 from dsDNA, the product complex of DNA strand exchange. Kinetic analysis of the budding yeast proteins revealed that the catalytic efficiency of the Rad54 ATPase was stimulated by partial filaments of wild-type and Rad51-K191R mutant protein on dsDNA, unambiguously demonstrating that the Rad54 ATPase activity is stimulated under these conditions. Experiments with Rad51-K191R as well as with wild-type Rad51-dsDNA filaments formed in the presence of ATP, ADP or ATP-gamma-S showed that efficient Rad51 turnover from dsDNA requires both the Rad51 ATPase and the Rad54 ATPase activities. The results with Rad51-K191R mutant protein also revealed an unexpected defect in binding to DNA. Once formed, Rad51-K191R-DNA filaments appeared normal upon electron microscopic inspection, but displayed significantly increased stability. These biochemical defects in the Rad51-K191R protein could lead to deficiencies in presynapsis (filament formation) and postsynapsis (filament disassembly) in vivo.

Show MeSH
Related in: MedlinePlus