E. coli Restriction Endonuclease

Caitlin Redak '17 and Ambert Sawaya '17


I. Introduction

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The E.coli restriction endonuclease is a type II restriction endonuclease, used to cleave double stranded DNA. It is often used experimentally, creating 4 nucleotide 5' overhangs that can be used for insertion of other DNA pieces. EcoR1 is a homodimer that exhibits sequence specific binding and cleavage. The specificity of EcoR1 is paramount to the molecule's presence in a cell. Without the specificity, the cell would run a risk of random cleavages that could be lethal. EcoR1 interacts with both the base pairs of DNA as well as the sugar-phosphate backbone. Intermolecular interactions are also crucial to the function of the protein. The catalytic activity of EcoR1 relies on a conformational change in both the DNA and the protein that aligns specific residues and bases as well as a divalent metal ion to create the active site.  

II. General Structure

EcoR1 is a 31 kDa homodimer consisting of 261 residues and a divalent metal . EcoR1 has two catalytic sites, separated by about 20 angstroms. The basic structure of EcoR1 is 5 beta sheets surrounded by alpha helices. A central major kink is responsible for unwinding DNA. There is present an extended chain motif running through the backbone of DNA during recognition and binding, parallel to the backbone. This motif is part of a recognition motif of 4 alpha helices. 

III. DNA Recognition

DNA binding of ECOR1 is highly specified. The nucleotide sequence 5'-GAATTC-3' is recognized by the homodimer on either strand of the DNA. When the DNA is recognized by ECOR1 a conformational change is undergone resulting in residues and the nucleotides rearranging so that they are ready for reaction by the attacking nucleophile for cleavage.

IV. DNA Binding and Cleavage

Once the protein has aligned the catalytic site with the appropriate nucleotide sequence, the divalent Mg2+ ion interacts with six ligands in each homodimer. The ion associates with one carboxylate oxygen of Glu111 , two carboxylate oxygens of Asp 91 , the main-chain carbonyl of Ala 112 , the O1P of the scissile phosphate GpAA , and a water molecule. This water molecule acts as the attacking nucleophile. In order to coordinate DNA properly with the Mg2+ ion, the positively charged side chain of Lys113 makes a hydrogen bond with the O2P of the scissile phosphate in GpAA . As a result, the carboxylate of Glu111 moves so that the OE2 atom also coordinates with the Mg2+ ion. Once  this positioning has a occurred, a water molecule is set up for nucleophilic attack. It shares one free electron pair with the Mg2+ ion and another is shared to the scissile phosphate of GpAA. The final coordination complex and water cleave the DNA at each catalytic site and create "sticky" ends for recombination or insertion.

VI. References

Kim, Y., Choi, J., Grable, Love, Greene, P. J., P., Rosenberg, J.M. 1990. Refinement of EcoR1 Endonuclease Crystal Structure: A Revised Protein Chain Tracing. Science., New Series, Vol. 300 (Sept 14, 1900), pp. 1307-1309.

Kurpiewski, M.R., Engler, L.E., Wozniak, L.A., Kobylanska, A., Koziolkiewicz, M., Stec, W. J., Jen-Jacobsen, L. 2004. Mechanisms of Coupling between DNA Recognition Specificity and Catalysis in EcoR1 Endonuclease. Structure. 12: 1775-1768.

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