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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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The effects of nucleotide cofactor on Rad51-dsDNA filament disassembly promoted by Rad54. (A) Rad51-dsDNA filaments were assembled at 4 bp/Rad51 (1.5 μM Rad51, 6 μM dsDNA) for 15 min at 30°C, either in the presence of 80% ADP (1 mM ATP and 4 mM ADP) or absence of ADP (5 mM ATP). Addition of Rad54 (50 nM) and scavenger DNA (30 µM) initiated the protein–DNA filament disassembly. Protein-free, labeled DNA substrate (lane 1); Rad51-dsDNA filaments in the absence of Rad54 but with ADP (lanes 2–5), in the presence of Rad54 and ADP (lanes 6–9), in the absence of Rad54 and ADP (lanes 10–13), in the presence of Rad54 but absence of ADP (lanes 14–17). (B) Rad51-dsDNA filaments were assembled as in (A) either in the presence of 20% ADP (4 mM ATP and 1 mM ADP) or absence of ADP (5 mM ATP). Addition of Rad54 (50 nM) and scavenger DNA (30 µM) initiated the protein–DNA filament disassembly. Protein-free, labeled DNA substrate (lane 1); Rad51-dsDNA filaments in the absence of Rad54 but with ADP (lanes 2–5), in the presence of Rad54 and ADP (lanes 6–9), in the absence of Rad54 and ADP (lanes 10–13), in the presence of Rad54 but absence of ADP (lanes 14–17). (C) The data from (A) and (B) were quantified on a PhosphoImager. The accumulation of free DNA was taken as readout of Rad51 displacement activity by Rad54. Data points represent the average of at least three replicate experiments and error bars represent one standard deviation. (D) Rad51-dsDNA filaments were assembled as in (A), either in the presence of ATP-γ-S (50 µM) or ATP (50 µM). Addition of Rad54 (50 nM), scavenger DNA (30 µM) and ATP (5 mM) initiates the protein–DNA filament disassembly. Protein-free, labeled DNA substrate (lane 1); Rad51-dsDNA filaments in the presence of Rad54 and ATP-γ-S (lanes 2–5), in the absence of Rad54 but with ATP-γ-S (lanes 6–9), in the presence of Rad54 but absence of ATP-γ-S (lanes 10–13), in the absence of Rad54 or ATP-γ-S (lanes 14–17). (E) The data from (D) were quantified on a PhosphoImager. The accumulation of the band of free DNA was taken as readout of Rad51 displacement activity by Rad54. Data points represent the average of at least three replicate experiments, and error bars represent 1 SD.
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Figure 6: The effects of nucleotide cofactor on Rad51-dsDNA filament disassembly promoted by Rad54. (A) Rad51-dsDNA filaments were assembled at 4 bp/Rad51 (1.5 μM Rad51, 6 μM dsDNA) for 15 min at 30°C, either in the presence of 80% ADP (1 mM ATP and 4 mM ADP) or absence of ADP (5 mM ATP). Addition of Rad54 (50 nM) and scavenger DNA (30 µM) initiated the protein–DNA filament disassembly. Protein-free, labeled DNA substrate (lane 1); Rad51-dsDNA filaments in the absence of Rad54 but with ADP (lanes 2–5), in the presence of Rad54 and ADP (lanes 6–9), in the absence of Rad54 and ADP (lanes 10–13), in the presence of Rad54 but absence of ADP (lanes 14–17). (B) Rad51-dsDNA filaments were assembled as in (A) either in the presence of 20% ADP (4 mM ATP and 1 mM ADP) or absence of ADP (5 mM ATP). Addition of Rad54 (50 nM) and scavenger DNA (30 µM) initiated the protein–DNA filament disassembly. Protein-free, labeled DNA substrate (lane 1); Rad51-dsDNA filaments in the absence of Rad54 but with ADP (lanes 2–5), in the presence of Rad54 and ADP (lanes 6–9), in the absence of Rad54 and ADP (lanes 10–13), in the presence of Rad54 but absence of ADP (lanes 14–17). (C) The data from (A) and (B) were quantified on a PhosphoImager. The accumulation of free DNA was taken as readout of Rad51 displacement activity by Rad54. Data points represent the average of at least three replicate experiments and error bars represent one standard deviation. (D) Rad51-dsDNA filaments were assembled as in (A), either in the presence of ATP-γ-S (50 µM) or ATP (50 µM). Addition of Rad54 (50 nM), scavenger DNA (30 µM) and ATP (5 mM) initiates the protein–DNA filament disassembly. Protein-free, labeled DNA substrate (lane 1); Rad51-dsDNA filaments in the presence of Rad54 and ATP-γ-S (lanes 2–5), in the absence of Rad54 but with ATP-γ-S (lanes 6–9), in the presence of Rad54 but absence of ATP-γ-S (lanes 10–13), in the absence of Rad54 or ATP-γ-S (lanes 14–17). (E) The data from (D) were quantified on a PhosphoImager. The accumulation of the band of free DNA was taken as readout of Rad51 displacement activity by Rad54. Data points represent the average of at least three replicate experiments, and error bars represent 1 SD.

Mentions: Rad51-dsDNA filaments assembled in the presence of 20% ADP were disassembled with faster kinetics than those assembled exclusively in the presence of ATP (Figure 6B, lanes 6–9, lanes 14–17, Figure 6C). Rad51-dsDNA filaments assembled in the presence of 80% ADP were disassembled even faster (Figure 6A, lanes 6–9, lanes 14–17, Figure 6C). This difference is not due to the inherent instability of Rad51-dsDNA filament assembled in the presence of ADP, since in the absence of Rad54, the amount of free DNA did not differ in reactions with 0%, 20% and 80% ADP. Rather, the filaments containing ADP-bound subunits displayed slower disappearance of the saturated complexes (Figure 6A, lanes 2–5, lanes 10–13; Figure 6B, lanes 2–5, lanes 10–13, Figure 6C). In these reactions, the ATP regeneration system was supplemented after filament assembly but before Rad54 addition to ensure optimal Rad54 activity. Control experiments confirmed that the Rad54 ATPase activity remained unaffected by the presence of ADP under these conditions (data not shown). Together, these results suggest that Rad54 prefers disassembling filaments containing Rad51 subunits bound to ADP. The observed effect represents a lower estimate as possible exchange of Rad51 bound ADP by ATP may occur under these reaction conditions.Figure 6.


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)

The effects of nucleotide cofactor on Rad51-dsDNA filament disassembly promoted by Rad54. (A) Rad51-dsDNA filaments were assembled at 4 bp/Rad51 (1.5 μM Rad51, 6 μM dsDNA) for 15 min at 30°C, either in the presence of 80% ADP (1 mM ATP and 4 mM ADP) or absence of ADP (5 mM ATP). Addition of Rad54 (50 nM) and scavenger DNA (30 µM) initiated the protein–DNA filament disassembly. Protein-free, labeled DNA substrate (lane 1); Rad51-dsDNA filaments in the absence of Rad54 but with ADP (lanes 2–5), in the presence of Rad54 and ADP (lanes 6–9), in the absence of Rad54 and ADP (lanes 10–13), in the presence of Rad54 but absence of ADP (lanes 14–17). (B) Rad51-dsDNA filaments were assembled as in (A) either in the presence of 20% ADP (4 mM ATP and 1 mM ADP) or absence of ADP (5 mM ATP). Addition of Rad54 (50 nM) and scavenger DNA (30 µM) initiated the protein–DNA filament disassembly. Protein-free, labeled DNA substrate (lane 1); Rad51-dsDNA filaments in the absence of Rad54 but with ADP (lanes 2–5), in the presence of Rad54 and ADP (lanes 6–9), in the absence of Rad54 and ADP (lanes 10–13), in the presence of Rad54 but absence of ADP (lanes 14–17). (C) The data from (A) and (B) were quantified on a PhosphoImager. The accumulation of free DNA was taken as readout of Rad51 displacement activity by Rad54. Data points represent the average of at least three replicate experiments and error bars represent one standard deviation. (D) Rad51-dsDNA filaments were assembled as in (A), either in the presence of ATP-γ-S (50 µM) or ATP (50 µM). Addition of Rad54 (50 nM), scavenger DNA (30 µM) and ATP (5 mM) initiates the protein–DNA filament disassembly. Protein-free, labeled DNA substrate (lane 1); Rad51-dsDNA filaments in the presence of Rad54 and ATP-γ-S (lanes 2–5), in the absence of Rad54 but with ATP-γ-S (lanes 6–9), in the presence of Rad54 but absence of ATP-γ-S (lanes 10–13), in the absence of Rad54 or ATP-γ-S (lanes 14–17). (E) The data from (D) were quantified on a PhosphoImager. The accumulation of the band of free DNA was taken as readout of Rad51 displacement activity by Rad54. Data points represent the average of at least three replicate experiments, and error bars represent 1 SD.
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Figure 6: The effects of nucleotide cofactor on Rad51-dsDNA filament disassembly promoted by Rad54. (A) Rad51-dsDNA filaments were assembled at 4 bp/Rad51 (1.5 μM Rad51, 6 μM dsDNA) for 15 min at 30°C, either in the presence of 80% ADP (1 mM ATP and 4 mM ADP) or absence of ADP (5 mM ATP). Addition of Rad54 (50 nM) and scavenger DNA (30 µM) initiated the protein–DNA filament disassembly. Protein-free, labeled DNA substrate (lane 1); Rad51-dsDNA filaments in the absence of Rad54 but with ADP (lanes 2–5), in the presence of Rad54 and ADP (lanes 6–9), in the absence of Rad54 and ADP (lanes 10–13), in the presence of Rad54 but absence of ADP (lanes 14–17). (B) Rad51-dsDNA filaments were assembled as in (A) either in the presence of 20% ADP (4 mM ATP and 1 mM ADP) or absence of ADP (5 mM ATP). Addition of Rad54 (50 nM) and scavenger DNA (30 µM) initiated the protein–DNA filament disassembly. Protein-free, labeled DNA substrate (lane 1); Rad51-dsDNA filaments in the absence of Rad54 but with ADP (lanes 2–5), in the presence of Rad54 and ADP (lanes 6–9), in the absence of Rad54 and ADP (lanes 10–13), in the presence of Rad54 but absence of ADP (lanes 14–17). (C) The data from (A) and (B) were quantified on a PhosphoImager. The accumulation of free DNA was taken as readout of Rad51 displacement activity by Rad54. Data points represent the average of at least three replicate experiments and error bars represent one standard deviation. (D) Rad51-dsDNA filaments were assembled as in (A), either in the presence of ATP-γ-S (50 µM) or ATP (50 µM). Addition of Rad54 (50 nM), scavenger DNA (30 µM) and ATP (5 mM) initiates the protein–DNA filament disassembly. Protein-free, labeled DNA substrate (lane 1); Rad51-dsDNA filaments in the presence of Rad54 and ATP-γ-S (lanes 2–5), in the absence of Rad54 but with ATP-γ-S (lanes 6–9), in the presence of Rad54 but absence of ATP-γ-S (lanes 10–13), in the absence of Rad54 or ATP-γ-S (lanes 14–17). (E) The data from (D) were quantified on a PhosphoImager. The accumulation of the band of free DNA was taken as readout of Rad51 displacement activity by Rad54. Data points represent the average of at least three replicate experiments, and error bars represent 1 SD.
Mentions: Rad51-dsDNA filaments assembled in the presence of 20% ADP were disassembled with faster kinetics than those assembled exclusively in the presence of ATP (Figure 6B, lanes 6–9, lanes 14–17, Figure 6C). Rad51-dsDNA filaments assembled in the presence of 80% ADP were disassembled even faster (Figure 6A, lanes 6–9, lanes 14–17, Figure 6C). This difference is not due to the inherent instability of Rad51-dsDNA filament assembled in the presence of ADP, since in the absence of Rad54, the amount of free DNA did not differ in reactions with 0%, 20% and 80% ADP. Rather, the filaments containing ADP-bound subunits displayed slower disappearance of the saturated complexes (Figure 6A, lanes 2–5, lanes 10–13; Figure 6B, lanes 2–5, lanes 10–13, Figure 6C). In these reactions, the ATP regeneration system was supplemented after filament assembly but before Rad54 addition to ensure optimal Rad54 activity. Control experiments confirmed that the Rad54 ATPase activity remained unaffected by the presence of ADP under these conditions (data not shown). Together, these results suggest that Rad54 prefers disassembling filaments containing Rad51 subunits bound to ADP. The observed effect represents a lower estimate as possible exchange of Rad51 bound ADP by ATP may occur under these reaction conditions.Figure 6.

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