User:Chase Haven/Sandbox 1
From Proteopedia
(New page: ==Rad51== ===Homologous Recombination=== The process of homologous recombination is essential for genomic stability through the high-fidelity repair of DNA double stranded breaks. The re...) |
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| - | ==Rad51== | + | ===Rad51=== |
| - | + | ==Homologous Recombination== | |
The process of homologous recombination is essential for genomic stability through the high-fidelity repair of DNA double stranded breaks. The recombination event is orchestrated by a family of enzymes called recombinases, which assemble into presynaptic filaments on single stranded DNA. The recombinases then perform a homology search, initiate strand invasion, resulting in homologous recombination and resolution of the double stranded break. Humans and yeast share the evolutionarily related recombinase Rad51, a homolog of the well-documented prokaryotic RecA protein. | The process of homologous recombination is essential for genomic stability through the high-fidelity repair of DNA double stranded breaks. The recombination event is orchestrated by a family of enzymes called recombinases, which assemble into presynaptic filaments on single stranded DNA. The recombinases then perform a homology search, initiate strand invasion, resulting in homologous recombination and resolution of the double stranded break. Humans and yeast share the evolutionarily related recombinase Rad51, a homolog of the well-documented prokaryotic RecA protein. | ||
| - | ''S. cerevisiae'' Rad51 is a 43 kDa protein that shows a remarkable degree of conservation with its human homolog. They share a core ATPase domain as well as an additional N-terminal domain (which is not observed in the bacterial RecA), although they lack a C-terminal extension characteristic of RecA. Coordination of the DNA binding properties of recombinase protomers directs their assembly into the functional unit of homologous recombination, the presynaptic filament, through nucleotide binding and hydrolysis | + | ''S. cerevisiae'' Rad51 is a 43 kDa protein that shows a remarkable degree of conservation with its human homolog. They share a core ATPase domain as well as an additional N-terminal domain (which is not observed in the bacterial RecA), although they lack a C-terminal extension characteristic of RecA. Coordination of the DNA binding properties of recombinase protomers directs their assembly into the functional unit of homologous recombination, the presynaptic filament, through nucleotide binding and hydrolysis as well as interactions with recombination mediator and auxiliary proteins. |
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| + | ==DNA Binding Properties== | ||
| + | As Rad51 comprises the paradigm of eukaryotic DNA strand exchange, extensive work has gone into characterizing its very unique DNA binding properties. It has been shown to bind ssDNA and dsDNA, each with two distinct modes. | ||
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| + | =====ssDNA Binding Modes===== | ||
| + | At pH 7.5 Rad51 monomers assemble onto ssDNA in the presence of ATP and Mg2+ ions. This occurs with an apparent stoichiometric ratio of 1 protein molecule to 4 nucleotides. This binding capability is lost in the absence of ATP, which contrasts with RecA’s ability to retain ssDNA binding capabilities without ATP. Similarly, pre-incubation of Rad51 with TRIS acetate prevents ssDNA binding if no ATP is present (due to the sequestering of Mg2+). Addition of ATP before this incubation prevents this inactivation. However, addition of ATP subsequent to incubation with TRIS acetate is unable to rescue any DNA binding ability. This signifies the necessity of the nucleotide cofactor with regards to its protective role in preserving Rad51’s ssDNA binding capabilities and preventing inactivation, which is believed to occur due to protein aggregation. | ||
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| + | The aforementioned binding characteristics are observed over a pH range of 6.8-8.5. However, at a pH lower than 6.8, the binding of ATP and presence of Mg2+ is no longer required. Interestingly, at these acidic pH values presynaptic filament assembly occurs in a different modality. Instead of the 4 nucleotides per Rad51 monomer, as observed at pH 7.5 with ATP and Mg2+, this observed binding mode occurs with a stoichiometry of either 6 or 7 nucleotides per monomer at pH 6.8 or 9 nucleotides per monomer at pH 6.2. | ||
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| + | The stability of these protein-DNA interactions can be evaluated by their resistance to disruption by addition of NaCl. The salt titration midpoint for the modes observed at pH 6.8 and 6.2 are 60 mM and 110 mM NaCl, respectively. Conversely, the salt titration midpoint for the binding mode observed at pH 7.5 in the presence of ATP and Mg2+ is 550 mM NaCl. This demonstrates that decreasing pH stabilizes the Rad51-ssDNA interaction when no nucleotide cofactor or Mg2+ is present, but also further conveyed the importance of ATP to proper filament assembly at pH values closer to physiological levels. Thus Rad51 exhibits two distinct and non-inter-convertible binding modes for ssDNA: a highly stable ATP-dependent one, and a weaker, ATP-independent manner. | ||
Revision as of 18:51, 14 May 2012
Contents |
Rad51
Homologous Recombination
The process of homologous recombination is essential for genomic stability through the high-fidelity repair of DNA double stranded breaks. The recombination event is orchestrated by a family of enzymes called recombinases, which assemble into presynaptic filaments on single stranded DNA. The recombinases then perform a homology search, initiate strand invasion, resulting in homologous recombination and resolution of the double stranded break. Humans and yeast share the evolutionarily related recombinase Rad51, a homolog of the well-documented prokaryotic RecA protein.
S. cerevisiae Rad51 is a 43 kDa protein that shows a remarkable degree of conservation with its human homolog. They share a core ATPase domain as well as an additional N-terminal domain (which is not observed in the bacterial RecA), although they lack a C-terminal extension characteristic of RecA. Coordination of the DNA binding properties of recombinase protomers directs their assembly into the functional unit of homologous recombination, the presynaptic filament, through nucleotide binding and hydrolysis as well as interactions with recombination mediator and auxiliary proteins.
DNA Binding Properties
As Rad51 comprises the paradigm of eukaryotic DNA strand exchange, extensive work has gone into characterizing its very unique DNA binding properties. It has been shown to bind ssDNA and dsDNA, each with two distinct modes.
ssDNA Binding Modes
At pH 7.5 Rad51 monomers assemble onto ssDNA in the presence of ATP and Mg2+ ions. This occurs with an apparent stoichiometric ratio of 1 protein molecule to 4 nucleotides. This binding capability is lost in the absence of ATP, which contrasts with RecA’s ability to retain ssDNA binding capabilities without ATP. Similarly, pre-incubation of Rad51 with TRIS acetate prevents ssDNA binding if no ATP is present (due to the sequestering of Mg2+). Addition of ATP before this incubation prevents this inactivation. However, addition of ATP subsequent to incubation with TRIS acetate is unable to rescue any DNA binding ability. This signifies the necessity of the nucleotide cofactor with regards to its protective role in preserving Rad51’s ssDNA binding capabilities and preventing inactivation, which is believed to occur due to protein aggregation.
The aforementioned binding characteristics are observed over a pH range of 6.8-8.5. However, at a pH lower than 6.8, the binding of ATP and presence of Mg2+ is no longer required. Interestingly, at these acidic pH values presynaptic filament assembly occurs in a different modality. Instead of the 4 nucleotides per Rad51 monomer, as observed at pH 7.5 with ATP and Mg2+, this observed binding mode occurs with a stoichiometry of either 6 or 7 nucleotides per monomer at pH 6.8 or 9 nucleotides per monomer at pH 6.2.
The stability of these protein-DNA interactions can be evaluated by their resistance to disruption by addition of NaCl. The salt titration midpoint for the modes observed at pH 6.8 and 6.2 are 60 mM and 110 mM NaCl, respectively. Conversely, the salt titration midpoint for the binding mode observed at pH 7.5 in the presence of ATP and Mg2+ is 550 mM NaCl. This demonstrates that decreasing pH stabilizes the Rad51-ssDNA interaction when no nucleotide cofactor or Mg2+ is present, but also further conveyed the importance of ATP to proper filament assembly at pH values closer to physiological levels. Thus Rad51 exhibits two distinct and non-inter-convertible binding modes for ssDNA: a highly stable ATP-dependent one, and a weaker, ATP-independent manner.
