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EcoRV endonuclease
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Introduction
EcoRV endonuclease is a type II restriction enzyme, or restriction endonuclease, found in e. coli bacteria. The main biological function of restriction endonucleases is to protect the cell's genome against any foreign DNA. Restriction enzymes recognize and cleave specific sequences of DNA. In the figure to the right, the enzyme is shown with the AAAGATATCTT. A type II restriction enzymes cleave at a short distance from the recognition site and often use Mg(2+) as a cofactor, as does this enzyme. They are commonly found in bacteria and shared structural features indicate that they are evolutionarily related. Type II endonucleases have been the site of much research because of applications in gene analysis and cloning and because they are great at modeling protein-DNA interactions.
Specifically, EcoRV is an orthodox restriction endonuclease. This means that the DNA sequence recognized is palindromic, meaning each strand contains the same sequence. The DNA duplex is cleaved at the phosphodiester bond located at 5'-GAT*ATC-3'. The other DNA strand will also be cleaved in the same location, producing blunt ends. This is relatively unique among restriction enzymes, as many cleave each DNA stand at a different location, leaving what are known as sticky ends. Cleavage occurs by the breaking of the bond between a 3' oxygen and the phosphorus by nucleophilic attack by water. The acidic residues Asp74 and Asp90 are located near the phosphodiester group and provide ligands for the activating Mg(2+) cofactor.
The steps of DNA cleavage are as follows. EcoRV binds to the DNA without specificity, which is followed by a diffusional walking "search" down the DNA molecule. If the protein encounters its recognition site, conformational changes occur in the enzyme-DNA complex. The binding to a section of DNA is looser, and when the protein encounters the of DNA it tightens and bends the DNA strands approximately 50 degrees. The DNA is cleaved and released from the enzyme.
(I'll delete these before the presentation, its just easier to work with them all in one spot)
Very interesting! A few suggestions: Tell us and use green scenes to show us which part of the DNA sequence (GATATC) is the recognition site and where is the cleavage site. Use color in the text to make it easier to quickly see your points: for example color the text and the DNA molecule red for the recognition sequence, then use color in both the text and structure to highlight the cleavage site.
If Mg2+ is present show it to us (space fill) and color "Mg" in the text the same color as in the structure.
If Mg is not present, is this to keep the enzyme from cleaving the DNA? That would be an interesting point to comment on.
You could try coloring the DNA and text to make the palindrome clear.
If you want to describe the active site, use colored text and a green scene to show us your points (or you could leave this to another section, or help with that section).
What are the structures shown in your non-specific and correct sequence green scenes? Are these different pdb file? Please include the pdb codes. You make very interesting points about the binding, but need better green scenes to explain this change (e.g. show the DNA bend and other conformational changes). I suggest passing these off to the Additional Features section, which currently has no green scenes and could make improved versions of these and fully explain the change.
I bet you could make a cooler caption for the molecular playground, about scissors for cutting DNA and cloning... but keep it similar length or shorter.
Length of your section is fine (about the length of jmol window): do not increase.
Overall Structure
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EcoRV endonuclease is functional as a dimer consisting of two monomers; both monomers depicted are shown in green or purple in a U shape. Each monomer, monomer A shown and monomer B shown , consists of 244 amino acids arranged in alpha/beta secondary structures, but the monomers are not identical.
Monomer B, previously shown in green, has 9 alpha helices, shown , in pink and 11 beta strands, shown , in blue. The beta strands form 3 beta sheets of various sizes, both parallel and anti parallel. The is a mixture of parallel and anti-parallel strands. The light purple and dark purple strands make up two anti-parallel sheets. The connection between the dark and light purple beta sheets is parallel. Depicted is a short triple-stranded antiparallel sheet that helps form the top side of each monomer. Opposite this beta sheet,shown , on the bottom of the enzyme, is another triple strand anti-parallel beta sheet.
Monomer A, previously shown in purple has 10 alpha helices, shown , in pink and 10 beta stands, shown , in blue. The differences between monomer A and monomer B lies only in the connection between the two monomers. At the point of connection a 5 strand beta sheet, made of 3 stands from monomer B and 2 strands from monomer A, this beta sheet assists in the structure and stability of the dimer as a whole. Shown , the green beta sheet is from monomer A and the purple beta sheet is from monomer B. Once you , you can see how these two anti-parallel beta sheets form one anti-parallel beta sheet connecting the two monomers at the bottom of the U shape. Monomer A also has an extra alpha helix, residue number 144-150, shown . For visual reference the monomer A and B connecting beta sheet is shown in dark purple (A beta strands) and dark green (B beta strands).
There are two structural sub-domains. The first, called the , is rather small and forms most of the dimer interface, residue numbers 19-32 and 150-160. The second is called the , residue numbers 2-18 38-140 and 167-243. The remaining segments of amino acids make up a the two sub-domains, residue numbers 33-37,141-149, and 161-165.
Good job making green scenes to illustrate your points. Your section is a bit repetitious and too long (should be about the length of jmol window): walking through the structure of each as you do comes out boring. Use color in the text to make your points more easily and concisely -- e.g. color "monomer B" the same green color. Try a more concise overview of what's in common, shown in Monomer B, and using color in the text -- e.g. the same color blue for the words beta strands and the strands in the green scene. Your goal is to say it in the shortest and most interesting way. omit the description and green scenes showing what's similar, which just repeats.
I'm guessing that monomers are identical sequence but not identical structure? Clarify this. An interesting part of what you've shown us is the structure stabilizing the dimer. Again use text color to communicate this more clearly. Also, the extra alpha helix in monomer A looks interesting: it has an arrow, so is it partly beta? Is it the same residue numbers that make the 3rd beta strand in monomer B? It almost looks like that 3rd beta strand is there in both monomers, but the stretch before it is helical in one case but not the other. You dark/light purple/green were not clear.
Binding Interactions
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DNA recognition sites on the EcoRV molecule, called R-loops, bind to the major grooves of the double stranded DNA at its recognition sequence GATATC by hydrogen bonding. This enzyme is a type II restriction endonuclease, which means this enzyme cleaves the DNA recognition sequence at the center (between the T and A base pairs). These hydrogen bonds makes the DNA form a kinked conformation that is later stabilized by the addition of the Mg2+ ion. The Mg2+ ion is a catalyst that causes the DNA to shift in a way that increases the rate necessary for DNA cleavage.
The Mg2+ binding site is formed when ionic interactions cause the slightly acidic Asp90 residue and the slightly negatively charged scissile phosphodiester group to approach each other. This allows the Mg2+ ion to bind to this enzyme, also with ionic interactions between the positively charged Mg2+ and the partially negative charged oxygen atoms from multiple molecules. These molecules that bind to the Mg2+ ion are the carboxylate oxygen atoms from the Asp74 and Asp90 residues, the nonesterified oxygen from the scissile phosphodiester group, and three additional oxygen atoms from three water molecules. These six ionic bonds form an octahedral shape in the active site of this enzyme.
These six ionic interactions all have about the same binding distance except for one bond between the oxygen from the Asp74 residue and the Mg2+ ion that is significantly longer. The five similar bond lengths are all about 2.08 Å, but the bond between Mg2+ and the Asp74 oxygen spans a distance of 2.9 Å. This is noted because the Asp90 and scissile phosphodiester molecules that bind to this Mg2+ ion change their bonding interactions with hydrogen to accommodate the addition of the Mg2+ ion. The Asp74 residue maintains its hydrogen bond interactions on its side chain with the main chain of the Ile91 residue and the water molecule, which is why it keeps a greater distance between itself and the Mg2+ ion.
You need to make green scenes to illustrate your points! You need to weave your scenes into the text, using colored text to help the reader easily see your points in the figure. Your scenes should fit with what you are trying to show. For instance, in your DNA binding scene it's not clear why you chose the color scheme you did (color should help you make one of your points, not just show us alpha vs beta structure) or why you included some side chains. Instead make multiple green scenes that each show a point you make in the text: the kink in the DNA, the Mg2+ binding site with its Asp74 and Asp90 and other ligands, the bond distances you mention can be shown with distance markers, etc. Text should not be any longer. Make a caption.
Additional Features
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As a prerequisite for efficient target site recognition, the enzyme is capable of nonspecific binding with the DNA phosphate backbone. Specific binding however is the process of recognition and is an interplay between interaction with the bases of the recognition sequence as well as indirect interaction with the respective phosphate backbone. Recognition precedes conformational changes in both DNA and the protein, thereby bending the DNA and activating the catalytic centers.
(working on green scenes and details of both types of interaction)
Don't tell us misc facts, instead focus on a few points you can tell us and illustrate with green scenes. You need to weave your scenes into the text, using colored text to help the reader easily see your points in the figure. Your scenes should fit with what you are trying to show. For instance, you could focus on the protein-induced DNA conformational change, and make green scenes that each illustrate what th text tells us: bending, unstacking, etc. You could also elaborate on the recognition of the specific sequence: wok this out with Jesse who started to talk about this so you're not repeating each other. Jesse could keep his short intro to this and you elaborate or you could move these points to your section. Add names if needed to share credit. Text length is OK -- keep it about the length of the jmol window. Make a caption.
Credits
Introduction - Jesse
Overall Structure - Nicole
DNA Binding - Julia
Additional Features - Sam
References
1. Kostrewa D, Winkler FK. Mg2+ binding to the active site of EcoRV endonuclease: a crystallographic study of complexes with substrate and product DNA at 2 A resolution. Biochemistry. 1995 Jan 17;34(2):683-96. PMID:7819264
2. Berg, J. Biochemistry, 7th edition.
3. Winkler, Fritz K. The crystal structure of EcoRV endonuclease and of its complexes with cognate and non-cognate DNA fragments. The EMBO Journal 12, p1781-1795. 1993
4. Pingoud A., Jeltsch A. Structure and function of type II restriction endonucleases. Nucleic Acids Research. 29 (18)3705–3727.
