Sandbox 666: Difference between revisions

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The function of ''Eco''RI is to cleave infectious DNA before it is methylated or before it begins to cause damage in the bacterial cell. That’s why ''Eco''RI is able to bind non-specifically anywhere to the DNA and continuously scans the DNA in a linear diffusion process with an approximate rate of 7*~~106~~ bp.s-1. In this way, the protein targets very quickly its specific sequences and any recognition sites is forgotten. This linear diffusion is due to electrostatic interactions; thence the DNA is trapped by ''Eco''RI but can move easily. Indeed during non-specific binding, there is no direct interaction with the bases of the DNA but there are only five interactions between amino acid residues from each subunit and the DNA phosphate groups. Moreover around 110 water molecules are located into this complex.

The function of ''Eco''RI is to cleave infectious DNA before it is methylated or before it begins to cause damage in the bacterial cell. That’s why ''Eco''RI is able to bind non-specifically anywhere to the DNA and continuously scans the DNA in a linear diffusion process with an approximate rate of 7*106 bp.s-1. In this way, the protein targets very quickly its specific sequences and any recognition sites is forgotten. This linear diffusion is due to electrostatic interactions; thence the DNA is trapped by ''Eco''RI but can move easily. Indeed during non-specific binding, there is no direct interaction with the bases of the DNA but there are only five interactions between amino acid residues from each subunit and the DNA phosphate groups. Moreover around 110 water molecules are located into this complex.

It was observed that the enzyme makes pauses when the site is very similar to the recognition site (affinity of the enzyme to these sites). When one of these sites or a methylated site are cut, the reactional process is very slow and takes place only in one strand of the DNA, so the cellular DNA ligase can repair the nicked DNA.

During the formation of the specific complex, the majority of the water molecules and ions are released and more and more direct interactions between the enzyme and the DNA are formed: some water molecules are immobilized between the enzyme and the DNA and formed hydrogen bounds, whose are important for the recognition and catalysis. Metal divalent ions, ~~Mg2~~+, play also an essential role in the recognition process. The two homodimeres of ~~EcoRI~~ cooperate in the binding and cleavage of their substrate and are symmetrically bound to the DNA. The specific complex formation leads to conformational changes of the protein and the DNA, thus catalytic centers of the both subunits are activated and the both DNA strands are simultaneously cleaved.

During the formation of the specific complex, the majority of the water molecules and ions are released and more and more direct interactions between the enzyme and the DNA are formed: some water molecules are immobilized between the enzyme and the DNA and formed hydrogen bounds, whose are important for the recognition and catalysis. Metal divalent ions, Mg2+, play also an essential role in the recognition process. The two homodimeres of ''Eco''RI cooperate in the binding and cleavage of their substrate and are symmetrically bound to the DNA. The specific complex formation leads to conformational changes of the protein and the DNA, thus catalytic centers of the both subunits are activated and the both DNA strands are simultaneously cleaved.

Specific binding bend the DNA by about 12° and involve interactions between ''Eco''RI and DNA bases and phosphodiester backbones over approximately 10-12 bp. Moreover the residues involve to binding could participate at the catalysis. The number of interactions between ''Eco''RI and the recognition sequence is maximal.

The restriction enzymes are highly specific and highly cooperative.<ref name="A" />

Sandbox 666: Difference between revisions

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-The function of ''Eco''RI is to cleave infectious DNA before it is methylated or before it begins to cause damage in the bacterial cell. That’s why ''Eco''RI is able to bind non-specifically anywhere to the DNA and continuously scans the DNA in a linear diffusion process with an approximate rate of 7*106 bp.s-1. In this way, the protein targets very quickly its specific sequences and any recognition sites is forgotten.  This linear diffusion is due to electrostatic interactions; thence the DNA is trapped by ''Eco''RI but can move easily. Indeed during non-specific binding, there is no direct interaction with the bases of the DNA but there are only five interactions between amino acid residues from each subunit and the DNA phosphate groups. Moreover around 110 water molecules are located into this complex.
+The function of ''Eco''RI is to cleave infectious DNA before it is methylated or before it begins to cause damage in the bacterial cell. That’s why ''Eco''RI is able to bind non-specifically anywhere to the DNA and continuously scans the DNA in a linear diffusion process with an approximate rate of 7*10<sup>6</sup> bp.s<sup>-1</sup>. In this way, the protein targets very quickly its specific sequences and any recognition sites is forgotten.  This linear diffusion is due to electrostatic interactions; thence the DNA is trapped by ''Eco''RI but can move easily. Indeed during non-specific binding, there is no direct interaction with the bases of the DNA but there are only five interactions between amino acid residues from each subunit and the DNA phosphate groups. Moreover around 110 water molecules are located into this complex.
 It was observed that the enzyme makes pauses when the site is very similar to the recognition site (affinity of the enzyme to these sites). When one of these sites or a methylated site are cut, the reactional process is very slow and takes place only in one strand of the DNA, so the cellular DNA ligase can repair the nicked DNA.
-During the formation of the specific complex, the majority of the water molecules and ions are released and more and more direct interactions between the enzyme and the DNA are formed: some water molecules are immobilized between the enzyme and the DNA and formed hydrogen bounds, whose are important for the recognition and catalysis.  Metal divalent ions, Mg2+, play also an essential role in the recognition process. The two homodimeres of EcoRI cooperate in the binding and cleavage of their substrate and are symmetrically bound to the DNA. The specific complex formation leads to conformational changes of the protein and the DNA, thus catalytic centers of the both subunits are activated and the both DNA strands are simultaneously cleaved.
+During the formation of the specific complex, the majority of the water molecules and ions are released and more and more direct interactions between the enzyme and the DNA are formed: some water molecules are immobilized between the enzyme and the DNA and formed hydrogen bounds, whose are important for the recognition and catalysis. Metal divalent ions, Mg<sup>2+</sup>, play also an essential role in the recognition process. The two homodimeres of ''Eco''RI cooperate in the binding and cleavage of their substrate and are symmetrically bound to the DNA. The specific complex formation leads to conformational changes of the protein and the DNA, thus catalytic centers of the both subunits are activated and the both DNA strands are simultaneously cleaved.
 Specific binding bend the DNA by about 12° and involve interactions between ''Eco''RI and DNA bases and phosphodiester backbones over approximately 10-12 bp. Moreover the residues involve to binding could participate at the catalysis. The number of interactions between ''Eco''RI and the recognition sequence is maximal.
 The restriction enzymes are highly specific and highly cooperative.<ref name="A" />