E. coli Restriction
Caitlin Redak '17 and Ambert Sawaya '17
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
A central major kink is responsible for unwinding DNA. There is
present an extended
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
the main-chain carbonyl of Ala
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.
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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