Sandbox 666: Difference between revisions

   

''Eco''RI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. ''Eco''RI has been extracted from strain R ''Escherichia coli'', a common bacterium, which populates the intestine of mammalians.  In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses) in both strands. Bacterial DNA is protected by a specific methylation of ''Eco''RI recognition sequences.

== Reaction ==

    

''Eco''RI is composed of two homodimers, so it has two identical subunits (Representation of one <scene name='Sandbox_666/Monomer_structure/4'>subunit</scene>) of 31 kDa, but it is possible to have homotetramers at high concentrations. The constitutive monomers are 276 amino acids long. ''Eco''RI and all the other restriction enzymes show a common structural core, which is a α/β domain. There are several non-contiguous structural elements, whose are involved in DNA recognition<ref name="A">Recognition and cleavage of DNA by type-II restriction endonucleases, Pingoud A. & Jeltsch A. Eur.J.Biochem. 246,1-22 (1997)</ref>. The constitutive subunits of ''Eco''RI are organized into a single α/β domain (five strands <scene name='Sandbox_666/B_sheet/1'>β</scene> sheet, which is surrounded  by <scene name='Sandbox_666/Helix/1'>two α helices</scene> ). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others<ref name="B">Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing, Kim, Y.C.,  Grable, J.C.,  Love, R.,  Greene, P.J.,  Rosenberg, J.M., Journal: (1990) Science 249: 1307-1309</ref>. Four helices (two of each subunit) recognize the major groove and bring residues, whose interact with DNA bases and backbones.

In the old model, the N-terminal section of each subunit forms the inner arm, which wraps around the DNA molecule (the arm brings the DNA molecule to the catalytic cleft). In the recent model, the extended chain motif ('''Met¹³⁷ to Ala¹⁴²''') is a segment of the extended polypeptide chain, which runs through the major groove of the DNA, roughly parallel to the DNA backbone<ref name="B" /> and forms the specific contacts of the enzymes to the DNA. The outer arm is composed of two minor β strands linked together by a loop (the outer arm is 14 amino acids long, four of these amino acids belong to the loop).

    

The specific recognition of ''Eco''RI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition: '''Arg²⁰⁰, Glu¹⁴⁴ and Arg¹⁴⁵'''. These amino acids are shown in red <scene name='Sandbox_666/Grey_protein/3'>here</scene>. Each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. There are also two arms, whose establish contacts with DNA backbones outside the recognition sequences.

    

There is one ß-strand parallel to the DNA backbone, which contains amino acid residues essential for catalysis (e.g.: residues engaged in phosphate contacts<ref name="A" />). 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

Alfred Pingoud, Albert Jeltsch

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