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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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Purification and characterization of Rad51 proteins. (A) Saccharomyces cerevisiae Rad51 (1 µg, lane 1), Rad51-K191R (1 µg, lane 2) and Rad51-K191A (1 µg, lane 3) proteins were purified from budding yeast, analyzed by 10% SDS–PAGE and stained with Coomassie brilliant blue R250. (B) ATPase activity of Rad51 (dark bars), Rad51-K191R (gray bars) and Rad51-K191A (white bars) proteins. The ATPase activities of Rad51 proteins (2.5 µM) were measured either in the presence of ssDNA (30 µM, poly-dA) or absence of DNA by an NADH-coupled microplate photometric assay. ATPase activity is measured as the rate of NADH decomposition (molar molecules of NADH decomposed per minute by molar molecule of Rad51). All measurements were done in triplicate, and the error bars represent 1 SD. (C) α-32P-ATP binding by Rad51 proteins. The Rad51-ATP complexes were resolved by 10% SDS–PAGE, and the signal intensities were quantified with a PhosphoImager. The intensity of the Rad51 band at 1 µM ATP was defined as one unit, and the signals were normalized to this standard. (D) Data obtained from (C) and additional gels were fitted into a saturation-binding curves using PRISM software (Graphpad). (E) α-P32-8-Azido-ATP binding by Rad51 proteins. (F) Fitted saturation binding curves for data obtained from (E) and additional gels. Procedures were as in (C, D). For (C–F), the calculated Kd values are shown in Table 1. All measurements were done in triplicate and the error bars represent 1 SD. For (C, D), the positions of MW markers, BSA and the Rad51 proteins were determined by Coomassie staining and are indicated on the left and right sides.
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Figure 1: Purification and characterization of Rad51 proteins. (A) Saccharomyces cerevisiae Rad51 (1 µg, lane 1), Rad51-K191R (1 µg, lane 2) and Rad51-K191A (1 µg, lane 3) proteins were purified from budding yeast, analyzed by 10% SDS–PAGE and stained with Coomassie brilliant blue R250. (B) ATPase activity of Rad51 (dark bars), Rad51-K191R (gray bars) and Rad51-K191A (white bars) proteins. The ATPase activities of Rad51 proteins (2.5 µM) were measured either in the presence of ssDNA (30 µM, poly-dA) or absence of DNA by an NADH-coupled microplate photometric assay. ATPase activity is measured as the rate of NADH decomposition (molar molecules of NADH decomposed per minute by molar molecule of Rad51). All measurements were done in triplicate, and the error bars represent 1 SD. (C) α-32P-ATP binding by Rad51 proteins. The Rad51-ATP complexes were resolved by 10% SDS–PAGE, and the signal intensities were quantified with a PhosphoImager. The intensity of the Rad51 band at 1 µM ATP was defined as one unit, and the signals were normalized to this standard. (D) Data obtained from (C) and additional gels were fitted into a saturation-binding curves using PRISM software (Graphpad). (E) α-P32-8-Azido-ATP binding by Rad51 proteins. (F) Fitted saturation binding curves for data obtained from (E) and additional gels. Procedures were as in (C, D). For (C–F), the calculated Kd values are shown in Table 1. All measurements were done in triplicate and the error bars represent 1 SD. For (C, D), the positions of MW markers, BSA and the Rad51 proteins were determined by Coomassie staining and are indicated on the left and right sides.

Mentions: The Rad51 ATPase activity appears to be critical for its biological function (12,20,25–28). Yet, the purified S. cerevisiae Rad51-K191R (or human Rad51-K133R) protein can catalyze in vitro recombination similar to the wild-type proteins (14,27). Substitution of the Walker A box residue K191 to R in the S. cerevisiae Rad51 protein (and K133 in human Rad51) has been assumed to block ATP hydrolysis but leave ATP binding unaffected, whereas the K to A substitution is assumed to abolish ATP binding, based on previous data (17,65). These assumptions have not been experimentally tested for S. cerevisiae Rad51. To understand the function of ATP hydrolysis by Rad51, in particular during the disassembly of the Rad51-dsDNA product complex by Rad54, we purified Rad51-K191R and Rad51-K191A proteins from the cognate host to apparent homogeneity (Figure 1A).Figure 1.


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)

Purification and characterization of Rad51 proteins. (A) Saccharomyces cerevisiae Rad51 (1 µg, lane 1), Rad51-K191R (1 µg, lane 2) and Rad51-K191A (1 µg, lane 3) proteins were purified from budding yeast, analyzed by 10% SDS–PAGE and stained with Coomassie brilliant blue R250. (B) ATPase activity of Rad51 (dark bars), Rad51-K191R (gray bars) and Rad51-K191A (white bars) proteins. The ATPase activities of Rad51 proteins (2.5 µM) were measured either in the presence of ssDNA (30 µM, poly-dA) or absence of DNA by an NADH-coupled microplate photometric assay. ATPase activity is measured as the rate of NADH decomposition (molar molecules of NADH decomposed per minute by molar molecule of Rad51). All measurements were done in triplicate, and the error bars represent 1 SD. (C) α-32P-ATP binding by Rad51 proteins. The Rad51-ATP complexes were resolved by 10% SDS–PAGE, and the signal intensities were quantified with a PhosphoImager. The intensity of the Rad51 band at 1 µM ATP was defined as one unit, and the signals were normalized to this standard. (D) Data obtained from (C) and additional gels were fitted into a saturation-binding curves using PRISM software (Graphpad). (E) α-P32-8-Azido-ATP binding by Rad51 proteins. (F) Fitted saturation binding curves for data obtained from (E) and additional gels. Procedures were as in (C, D). For (C–F), the calculated Kd values are shown in Table 1. All measurements were done in triplicate and the error bars represent 1 SD. For (C, D), the positions of MW markers, BSA and the Rad51 proteins were determined by Coomassie staining and are indicated on the left and right sides.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 1: Purification and characterization of Rad51 proteins. (A) Saccharomyces cerevisiae Rad51 (1 µg, lane 1), Rad51-K191R (1 µg, lane 2) and Rad51-K191A (1 µg, lane 3) proteins were purified from budding yeast, analyzed by 10% SDS–PAGE and stained with Coomassie brilliant blue R250. (B) ATPase activity of Rad51 (dark bars), Rad51-K191R (gray bars) and Rad51-K191A (white bars) proteins. The ATPase activities of Rad51 proteins (2.5 µM) were measured either in the presence of ssDNA (30 µM, poly-dA) or absence of DNA by an NADH-coupled microplate photometric assay. ATPase activity is measured as the rate of NADH decomposition (molar molecules of NADH decomposed per minute by molar molecule of Rad51). All measurements were done in triplicate, and the error bars represent 1 SD. (C) α-32P-ATP binding by Rad51 proteins. The Rad51-ATP complexes were resolved by 10% SDS–PAGE, and the signal intensities were quantified with a PhosphoImager. The intensity of the Rad51 band at 1 µM ATP was defined as one unit, and the signals were normalized to this standard. (D) Data obtained from (C) and additional gels were fitted into a saturation-binding curves using PRISM software (Graphpad). (E) α-P32-8-Azido-ATP binding by Rad51 proteins. (F) Fitted saturation binding curves for data obtained from (E) and additional gels. Procedures were as in (C, D). For (C–F), the calculated Kd values are shown in Table 1. All measurements were done in triplicate and the error bars represent 1 SD. For (C, D), the positions of MW markers, BSA and the Rad51 proteins were determined by Coomassie staining and are indicated on the left and right sides.
Mentions: The Rad51 ATPase activity appears to be critical for its biological function (12,20,25–28). Yet, the purified S. cerevisiae Rad51-K191R (or human Rad51-K133R) protein can catalyze in vitro recombination similar to the wild-type proteins (14,27). Substitution of the Walker A box residue K191 to R in the S. cerevisiae Rad51 protein (and K133 in human Rad51) has been assumed to block ATP hydrolysis but leave ATP binding unaffected, whereas the K to A substitution is assumed to abolish ATP binding, based on previous data (17,65). These assumptions have not been experimentally tested for S. cerevisiae Rad51. To understand the function of ATP hydrolysis by Rad51, in particular during the disassembly of the Rad51-dsDNA product complex by Rad54, we purified Rad51-K191R and Rad51-K191A proteins from the cognate host to apparent homogeneity (Figure 1A).Figure 1.

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