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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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Related in: MedlinePlus

Topological assay of Rad51-dsDNA filament disassembly by Rad54. (A) Scheme of the assay. Nucleoprotein filaments were formed by incubating Rad51 or Rad51-K191R protein with the supercoiled DNA substrate. Binding by Rad51 unwinds the dsDNA and leads to the introduction of compensatory positive scs (+). Relaxation of these scs by topoisomerase I followed by deproteinization results in highly negatively supercoiled DNA, so called form X DNA (x, top). If Rad51 is completely removed from the DNA substrate by Rad54 before the treatment of topoisomerase, the substrate will be converted to a fully relaxed species (re, bottom). If Rad51 was partially removed, it will be converted to species with varying degree of negative supercoiling (x, top; sc, middle). (B) ATPase activity of Rad51 is important for its dissociation from dsDNA promoted by Rad54. Rad51 or Rad51-K191R (7.5 µM) was pre-incubated with pUC19 dsDNA (30 µM) for 30 min at 23°C to form nucleoprotein filaments. Rad54 (0.375 µM) was added and incubated for 5 min, followed by the addition of scavenger DNA (63 µM PstI-linearized M13mp19 dsDNA), and incubated for 2 h at 23°C. Reactions were separated on a native 1.2% agarose gel without chloroquine in the first dimension followed by a second electrophoresis step with 4 µg/ml chloroquine. The positions of supercoiled (sc), relaxed (re), X form (x) and scavenger (scav) dsDNA are indicated in the first gel for the reaction with Rad51 protein.
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Figure 5: Topological assay of Rad51-dsDNA filament disassembly by Rad54. (A) Scheme of the assay. Nucleoprotein filaments were formed by incubating Rad51 or Rad51-K191R protein with the supercoiled DNA substrate. Binding by Rad51 unwinds the dsDNA and leads to the introduction of compensatory positive scs (+). Relaxation of these scs by topoisomerase I followed by deproteinization results in highly negatively supercoiled DNA, so called form X DNA (x, top). If Rad51 is completely removed from the DNA substrate by Rad54 before the treatment of topoisomerase, the substrate will be converted to a fully relaxed species (re, bottom). If Rad51 was partially removed, it will be converted to species with varying degree of negative supercoiling (x, top; sc, middle). (B) ATPase activity of Rad51 is important for its dissociation from dsDNA promoted by Rad54. Rad51 or Rad51-K191R (7.5 µM) was pre-incubated with pUC19 dsDNA (30 µM) for 30 min at 23°C to form nucleoprotein filaments. Rad54 (0.375 µM) was added and incubated for 5 min, followed by the addition of scavenger DNA (63 µM PstI-linearized M13mp19 dsDNA), and incubated for 2 h at 23°C. Reactions were separated on a native 1.2% agarose gel without chloroquine in the first dimension followed by a second electrophoresis step with 4 µg/ml chloroquine. The positions of supercoiled (sc), relaxed (re), X form (x) and scavenger (scav) dsDNA are indicated in the first gel for the reaction with Rad51 protein.

Mentions: Both, Rad51-K191R and Rad51-K191A proteins bound DNA in a nucleotide-dependent manner (data not shown). We utilized primarily electrophoretic analysis of glutaraldehyde-fixed Rad51-DNA complexes, a method normally used with RecA (19,68) and human Rad51 (28,69). We have verified in previous studies that the electrophoretic mobility of yeast Rad51-DNA complexes corresponds within the limits of resolution to filament saturation using electron microscopy, topological assays (Figure 5), and nuclease protection assays (41,61,62). An earlier analysis failed to detect Rad51-K191A binding to DNA (44), but using fixation prior to electrophoretic analysis stabilized the Rad51-K191A-filaments sufficiently to allow their visualization (data not shown). The Rad51-K191A-dsDNA filament displayed a significant defect in its interaction with Rad54 protein. Since the purpose of the study is to understand the interaction between the Rad51 and Rad54 proteins, we focused on the Rad51-K191R protein and its interaction with Rad54 for the further analysis.


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)

Topological assay of Rad51-dsDNA filament disassembly by Rad54. (A) Scheme of the assay. Nucleoprotein filaments were formed by incubating Rad51 or Rad51-K191R protein with the supercoiled DNA substrate. Binding by Rad51 unwinds the dsDNA and leads to the introduction of compensatory positive scs (+). Relaxation of these scs by topoisomerase I followed by deproteinization results in highly negatively supercoiled DNA, so called form X DNA (x, top). If Rad51 is completely removed from the DNA substrate by Rad54 before the treatment of topoisomerase, the substrate will be converted to a fully relaxed species (re, bottom). If Rad51 was partially removed, it will be converted to species with varying degree of negative supercoiling (x, top; sc, middle). (B) ATPase activity of Rad51 is important for its dissociation from dsDNA promoted by Rad54. Rad51 or Rad51-K191R (7.5 µM) was pre-incubated with pUC19 dsDNA (30 µM) for 30 min at 23°C to form nucleoprotein filaments. Rad54 (0.375 µM) was added and incubated for 5 min, followed by the addition of scavenger DNA (63 µM PstI-linearized M13mp19 dsDNA), and incubated for 2 h at 23°C. Reactions were separated on a native 1.2% agarose gel without chloroquine in the first dimension followed by a second electrophoresis step with 4 µg/ml chloroquine. The positions of supercoiled (sc), relaxed (re), X form (x) and scavenger (scav) dsDNA are indicated in the first gel for the reaction with Rad51 protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919488&req=5

Figure 5: Topological assay of Rad51-dsDNA filament disassembly by Rad54. (A) Scheme of the assay. Nucleoprotein filaments were formed by incubating Rad51 or Rad51-K191R protein with the supercoiled DNA substrate. Binding by Rad51 unwinds the dsDNA and leads to the introduction of compensatory positive scs (+). Relaxation of these scs by topoisomerase I followed by deproteinization results in highly negatively supercoiled DNA, so called form X DNA (x, top). If Rad51 is completely removed from the DNA substrate by Rad54 before the treatment of topoisomerase, the substrate will be converted to a fully relaxed species (re, bottom). If Rad51 was partially removed, it will be converted to species with varying degree of negative supercoiling (x, top; sc, middle). (B) ATPase activity of Rad51 is important for its dissociation from dsDNA promoted by Rad54. Rad51 or Rad51-K191R (7.5 µM) was pre-incubated with pUC19 dsDNA (30 µM) for 30 min at 23°C to form nucleoprotein filaments. Rad54 (0.375 µM) was added and incubated for 5 min, followed by the addition of scavenger DNA (63 µM PstI-linearized M13mp19 dsDNA), and incubated for 2 h at 23°C. Reactions were separated on a native 1.2% agarose gel without chloroquine in the first dimension followed by a second electrophoresis step with 4 µg/ml chloroquine. The positions of supercoiled (sc), relaxed (re), X form (x) and scavenger (scav) dsDNA are indicated in the first gel for the reaction with Rad51 protein.
Mentions: Both, Rad51-K191R and Rad51-K191A proteins bound DNA in a nucleotide-dependent manner (data not shown). We utilized primarily electrophoretic analysis of glutaraldehyde-fixed Rad51-DNA complexes, a method normally used with RecA (19,68) and human Rad51 (28,69). We have verified in previous studies that the electrophoretic mobility of yeast Rad51-DNA complexes corresponds within the limits of resolution to filament saturation using electron microscopy, topological assays (Figure 5), and nuclease protection assays (41,61,62). An earlier analysis failed to detect Rad51-K191A binding to DNA (44), but using fixation prior to electrophoretic analysis stabilized the Rad51-K191A-filaments sufficiently to allow their visualization (data not shown). The Rad51-K191A-dsDNA filament displayed a significant defect in its interaction with Rad54 protein. Since the purpose of the study is to understand the interaction between the Rad51 and Rad54 proteins, we focused on the Rad51-K191R protein and its interaction with Rad54 for the further analysis.

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