Sandbox 666: Difference between revisions

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The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…(D/E)XK motif. For ''Eco''RI,<scene name='Sandbox_666/Catalytic_core/3'>this catalytic sequence</scene> is PD91 …E111AK and the lysine residue is essential to the catalysis, but the proline residue is not important. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).

The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…(D/E)XK motif. For ''Eco''RI,<scene name='Sandbox_666/Catalytic_core/3'>this catalytic sequence</scene> is PD91 …E111AK and the lysine residue is essential to the catalysis, but the proline residue is not important. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).<ref>Structure and function of type II restriction endonucleases

~~[[Image:Catalytic 1ERI.png | thumb | The catalytic core of Eco RI with the target DNA|left|400px]]~~<ref>

Structure and function of type II restriction endonucleases

Alfred Pingoud, Albert Jeltsch

Nucleic Acids Res. 2001 September 15; 29(18): 3705–3727.

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There are several non-contiguous structural elements, which are involved in DNA recognition:

Four helix (two of each subunit) recognize the major groove and bring residues, whose interact with DNA bases and backbones

An extended peptide chain running through the major groove forms the specific contacts of the enzymes to the DNA.

-Four helix (two of each subunit) recognize the major groove and bring residues, whose interact with DNA bases and backbones

One ß-strand parallel to the DNA backbone, which contains amino acid residues essential for catalysis (e.g.: residues engaged in phosphate contacts)

Two arms, which establish contacts with DNA backbones outside the recognition sequences.

-An extended peptide chain running through the major groove forms the specific contacts of the enzymes to the DNA.

-One ß-strand parallel to the DNA backbone, which contains amino acid residues essential for catalysis (e.g.: residues engaged in phosphate contacts)

-Two arms, which establish contacts with DNA backbones outside the recognition sequences.

This binding by the major groove is due to the position of scissile phosphodiester bonds.

[[Image:Catalytic 1ERI.png | thumb | The catalytic core of Eco RI with the target DNA|left|400px]]

==Catalytic mechanism==

The function of EcoRI is to cleave infectious DNA before it is methylated or before it begins to cause damage in the bacterial cell. That’s why EcoRI 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 EcoRI 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.

Specific binding bend the DNA by about 12° and involve interactions between EcoRI 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 EcoRI and the recognition sequence is maximal.

The restriction enzymes are highly specific and highly cooperative.<ref>Recognition and cleavage of DNA by type-II restriction endonucleases, Pingoud A. & Jeltsch A. Eur.J.Biochem. 246,1-22 (1997)</ref>

== Application of EcoRI in molecular biology ==

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== References ==

<references/> ~~6. ↑ Recognition and cleavage of DNA by type-II restriction endonucleases, Pingoud A. & Jeltsch A. Eur.J.Biochem. 246,1-22 (1997)~~

== Contributor ==

Raphaël BILGER Virginie GROSBOILLOT

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@@ Line 26: / Line 26: @@
-The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…(D/E)XK motif. For ''Eco''RI,<scene name='Sandbox_666/Catalytic_core/3'>this catalytic sequence</scene> is PD<sup>91</sup> …E<sup>111</sup>AK and the lysine residue is essential to the catalysis, but the proline residue is not important. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).
+The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…(D/E)XK motif. For ''Eco''RI,<scene name='Sandbox_666/Catalytic_core/3'>this catalytic sequence</scene> is PD<sup>91</sup> …E<sup>111</sup>AK and the lysine residue is essential to the catalysis, but the proline residue is not important. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).<ref>Structure and function of type II restriction endonucleases
- [[Image:Catalytic 1ERI.png | thumb | The catalytic core of Eco RI with the target DNA|left|400px]]<ref>
-Structure and function of type II restriction endonucleases
 Alfred Pingoud, Albert Jeltsch
 Nucleic Acids Res. 2001 September 15; 29(18): 3705–3727.
@@ Line 34: / Line 32: @@
 There are several non-contiguous structural elements, which are involved in DNA recognition:
-    Four helix (two of each subunit) recognize the major groove and bring residues, whose interact with DNA bases and backbones
-    An extended peptide chain running through the major groove forms the specific contacts of the enzymes to the DNA.
+-Four helix (two of each subunit) recognize the major groove and bring residues, whose interact with DNA bases and backbones
-    One ß-strand parallel to the DNA backbone, which contains amino acid residues essential for catalysis (e.g.: residues engaged in phosphate contacts)
-    Two arms, which establish contacts with DNA backbones outside the recognition sequences.
+-An extended peptide chain running through the major groove forms the specific contacts of the enzymes to the DNA.
+-One ß-strand parallel to the DNA backbone, which contains amino acid residues essential for catalysis (e.g.: residues engaged in phosphate contacts)
+-Two arms, which establish contacts with DNA backbones outside the recognition sequences.
 This binding by the major groove is due to the position of scissile phosphodiester bonds.
+[[Image:Catalytic 1ERI.png | thumb | The catalytic core of Eco RI with the target DNA|left|400px]]
+==Catalytic mechanism==
+The function of EcoRI is to cleave infectious DNA before it is methylated or before it begins to cause damage in the bacterial cell. That’s why EcoRI 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 EcoRI 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.
+Specific binding bend the DNA by about 12° and involve interactions between EcoRI 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 EcoRI and the recognition sequence is maximal.
+The restriction enzymes are highly specific and highly cooperative.<ref>Recognition and cleavage of DNA by type-II restriction endonucleases, Pingoud A. & Jeltsch A. Eur.J.Biochem. 246,1-22 (1997)</ref>
 == Application of EcoRI in molecular biology ==
@@ Line 49: / Line 64: @@
 == References ==
-<references/>      6.  ↑ Recognition and cleavage of DNA by type-II restriction endonucleases, Pingoud A. & Jeltsch A. Eur.J.Biochem. 246,1-22 (1997)
+<references/>
 == Contributor ==
 Raphaël BILGER  Virginie GROSBOILLOT