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| <!-- DO NOT DELETE THE TEMPLATE LINE --> | | Ref<ref>PMID:13990617</ref> |
| {{Template:Sandbox Reserved Lynmarie Thompson}}
| | Ref<ref>PMID:4882249</ref> |
| <!-- INSERT YOUR SCENES AND TEXT BELOW THIS LINE --> | | Ref<ref>PMID:5884016</ref> |
| | | Ref<ref>PMID:11389934</ref> |
| =='''EcoRV endonuclease'''==
| | Ref<ref>PMID:12557186</ref> |
| | | Ref<ref>PMID: 5905118 </ref> |
| <Structure load='1rva' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' />
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| ===Introduction===
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| EcoRV endonuclease is a type II restriction enzyme found in e. coli bacteria. Restriction enzymes recognize and cleave specific sequences of DNA. In the figure to the right, the enzyme is shown with the <scene name='Sandbox_Reserved_428/Dna_sequence/1'>eleven base sequence</scene> 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. Type II restriction enzymes are commonly found in bacteria and shared structural features indicate that they are evolutionarily related.
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| The DNA duplex is cleaved at the phosphodiester bond located at GAT*ATC. Cleavage occurs by the breaking of the bond between a 3' oxygen and the phosphorus by nucleophilic attack by water.
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| <scene name='Sandbox_Reserved_428/Dna_sequence/1'>TextToBeDisplayed</scene> | |
| <scene name='Sandbox_Reserved_428/Dna_zoom/1'>DNA zoom</scene> | |
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| ===Overall Structure===
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| EcoRV endonuclease is functional as a dimer consisting of two monomers; both monomers depicted <scene name='Sandbox_Reserved_428/U-shaped_dimer/1'>here</scene> are shown in green or purple in a U shape. Each monomer, monomer A shown <scene name='Sandbox_Reserved_428/Monomera/1'>here</scene> and monomer B shown <scene name='Sandbox_Reserved_428/Monomer/1'>here</scene>, consists of 244 amino acids arranged in alpha/beta secondary structures, but the monomers are not identical.
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| Monomer A, previously shown in purple has 10 alpha helices, shown <scene name='Sandbox_Reserved_428/Alpha_helix_-_monomera/1'>here</scene>, in pink and 10 beta stands, shown <scene name='Sandbox_Reserved_428/Beta_sheets_-_monomera/1'>here</scene, in blue.
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| Monomer B, previously shown in green, has 9 alpha helices, shown <scene name='Sandbox_Reserved_428/Alpha_helix_-_monomer/1'>here</scene>, in pink and 11 beta strands, shown <scene name='Sandbox_Reserved_428/Beta_strands_-_monomer/1'>here</scene>, in blue. The beta strands form 3 beta sheets of various sizes, both parallel and anti parallel. The <scene name='Sandbox_Reserved_428/Largest_beta_sheet_-_monomer/1'>largest beta sheet</scene> 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 <scene name='Sandbox_Reserved_428/Tripleap_beta_sheets_-_monomer/1'>here</scene> is a short triple-stranded antiparallel sheet that helps form the top side of each monomer. Opposite this beta sheet,shown <scene name='Sandbox_Reserved_428/Apattachbeta_strands_-_monomer/4'>here</scene>, on the bottom of the enzyme, is another triple strand anti-parallel beta sheet. By forming a 5 strand beta sheet with the same stands on the other monomer, this beta sheet assists in the structure and stability of the dimer as a whole. Shown <scene name='Sandbox_Reserved_428/Beta_sheets_-_dimer/1'>here</scene>, the green beta sheet is from monomer A and the purple beta sheet is from monomer B. Once you <scene name='Sandbox_Reserved_428/Zoom_out_beta_sheets_-_dimer/1'>zoom out</scene>, 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.
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| ===Binding Interactions===
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| <scene name='Sandbox_Reserved_428/Binds_to_dna/1'>DNA Binding</scene> | |
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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.
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| 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.
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| 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.
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| ===Additional Features===
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| ===Credits===
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| Introduction - Jesse
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| Overall Structure - Nicole
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| Drug Binding Site - Julia
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| Additional Features - Sam
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| ===References===
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| <references/> | | <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
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| 2. Berg, J. ''Biochemistry,'' 7th edition.
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