EcoRl structure

Kim, Y, Grable, J. C., Love, R., Greene, P. J. and Rosenberg, J. M. (1990). Refinement of EcoRl endonuclease crystal structure a revised protein chain tracing. Science 249, 1307-1309. 

Fig. 2. Structure of the fusion vector pSV-VNPy2bA(CH2, CH3). The VNP and y2b genes are inserted in pSVgpt between the EcoRl and BamHl sites. The BamHl site is converted to Xhol by the addition of linkers B, Bgl l H, Hin WV, P, Pvu R, EcoRl, X, Xhol, E, immunoglobulin enhancer.

Nordland, T. M., Andersson, S., Nilsson, L., Rigler, R., Grasland, A., and McLaughlin, L. W. (1989). Structure and dynamics of a fluorescent DNA oligomer containing the EcoRl recognition sequence Fluorescence, molecular dynamics, and NMR studies. Biochemistry 28, 9095-9103. 

When EcoRl cuts the DNA at this site, it does so in a staggered fashion. Specifically, it cuts between the G and the first A on both strands. Cutting produces two DNA fragments with the following structure ... 

The role of the divalent metal ions present in natural phosphodiesterases became clear in bovine pancreatic deoxyribonuclease I (DNase I), the first endonuclease structure determined by X-ray crystallography. The nucleophilic attack of a water molecule activated by a histidine residue is facilitated by the interaction of a calcium ion with the phosphate group to be cleaved (291). Glutamic and aspartic residues involved in magnesium binding have been identified in the crystal structure of four type II restriction enzymes EcoRl (292), EcoRV (293), Pvull (294), and BamHl (295), as well as in that of the repair... 

The unusual conformation of the isolated EcoRl-PstI SV40 fragment. The findings reported above could reveal new information about the mechanism of binding of poly(ADP-ribose) polymerase to unusual secondary structures on DNA. DNA stmcture is known to be polymorphic (5, 6). Chemical and enzymatic probes (e.g. SI nuclease) are available to... 

Measurements of excimer or exciplex fluorescence can be used to determine whether a pair of macromolecules or two regions of a macromolecule are able to come in close contact during the lifetime of the excited state. Derivatives of pyrene that can be attached to various functional groups in proteins, lipids or polysaccharides lend themselves well to such studies [57-61]. In one application, the A-terminus of the EcoRl restriction endonuclease was labeled with N-(l-pyrenyl)iodoacetamide [62]. A broad excimer emission band at 480 nm indicated that the A-termini of two molecules come into close proximity when the protein dimerizes. The A-termini are essential for enzymatic activity but are too disordered to be seen in a crystal structure of the protein. 

EcoRl MTase (M EcoRl) is a member of the type II adenine-MTases whose structure and function have been relatively well characterized. It is a monomeric enzyme with a molecular mass of 38 kDa, an average size for type II MTases. M EcoRI recognizes the same hexanucleotide sequence as R EcoRI, and methylates the central adenine residues of both strands at the exocyclic N-6 amino group resulting in N -methyl-A (m A or " A) S -GA ATTC-S /S -CTT AAG-S (1). The MTase requires as substrates only AdoMet and DNA that is unmodified in at least one strand. 

The presence of a number of CIDNP-sensitive amino acids on the protein surface can enable the detection of conformational alterations resulting from single site mutations. This has been demonstrated for wild-type forms and various mutants of EcorL and of the sialidase of Clostridium perfringens [5, 7], Sialidase of C. perfringens is larded with CIDNP-responsive amino acid residues. Therefore, these residues have been used as valuable sensors in NMR and modelling studies in which CIDNP-spectra of the sialidase wild type and various mutants are compared and correlated with modelled structures. 

Figure 7. MD-derived energy minimum structures of wild-type EcorL and single-site mutants, a. wild-type EcorL b. mutant [Y229A]EcorL.

In addition to the above quaternary structures, two additional types of legume lectin dimers have been found in the GS-IV and ECorL lectins, which are both dimeric proteins [26]. The peanut lectin, which is a tetramer, has a unique structure composed of two GS-IV type dimers [27]. Such variation in quaternary structure of the legume lectins may be of significance to the role(s) of these lectins in the plant. 

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