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10  structures 50  species 1  interaction 52  sequences 1  architecture

Family: EcoRI (PF02963)

Summary: Restriction endonuclease EcoRI

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EcoRI Edit Wikipedia article

EcoRI
PDB 1qc9 EBI.jpg
The crystallographic structure of restriction endonuclease EcoRI at 3.3 a in the absence of DNA
Identifiers
Symbol EcoRI
Pfam PF02963
InterPro IPR004221
SCOP 1na6
SUPERFAMILY 1na6

EcoRI (pronounced, "eco R one") is an endonuclease enzyme isolated from strains of E. coli, and is part of the restriction modification system.

In molecular biology it is used as a restriction enzyme. It creates sticky ends with 5' end overhangs. The nucleic acid sequence where the enzyme cuts is GAATTC, which, as the complementary sequence is CTTAAG, has rotational symmetry.

Structure

Primary structure

EcoRI contains the PD..D/EXK motif within its active site like many restriction endonucleases.

EcoRI crystal structure. Dimer bound to DNA (PDB 1ckq)

Tertiary and quaternary structure

The enzyme is a homodimer of a 31 kilodalton subunit consisting of one globular domain of the α/β architecture. Each subunit contains a loop which sticks out from the globular domain and wraps around the DNA when bound.[1][2]

EcoRI recognition site with cutting pattern indicated by a green line

EcoRI has been cocrystallized with the sequence it normally cuts. This crystal was used to solve the structure of the complex 1QPS. The solved crystal structure shows that the subunits of the enzyme homodimer interact with the DNA symmetrically.[1] In the complex, two α-helices from each subunit come together to form a four helix bundle.[3] On the interacting helices are residues Glu144 and Arg145, which interact together forming a crosstalk ring that is believed to allow the enzyme's two active sites to communicate.[4]

Uses

Restriction enzymes such as EcoRI are used in a wide variety of molecular genetics techniques including cloning, DNA screening and deleting sections of DNA in vitro. Restriction enzymes like EcoRI that generate sticky ends of DNA are often used to cut DNA prior to ligation, as the sticky ends make the ligation reaction more efficient. EcoRI can exhibit non site-specific cutting, known as star activity, depending on the conditions present in the reaction. Conditions that can induce star activity when using EcoRI include low salt concentration, high glycerol concentration, excessive amounts of enzyme present in the reaction, high pH and contamination with certain organic solvents.[5]

See also

References

  1. ^ a b Pingoud, A., Jeltsch, A. (2001). "Structure and function of type II restriction endonucleases". Nucl. Acids Res 29 (18): 3705–3727. doi:10.1093/nar/29.18.3705. PMC 55916. PMID 11557805. 
  2. ^ Kurpiewski, M. R., Engler, L. E., Wozniak, L. A., Kobylanska, A., Koziolkiewicz, M., Stec, W. J, Jen-Jacobson, L (2004). "Mechanisms of coupling between DNA recognition and catalysis in EcoRI endonucleases". Structure 12: 1775–1788. doi:10.1016/j.str.2004.07.016. PMID 15458627. 
  3. ^ Bitinaite, J., D. A. Wah, Aggarwal, A. K., Schildkraut, I. (1998). "FokI dimerization is required for DNA cleavage". Proc Natl Acad Sci U S A 95 (18): 10570–5. doi:10.1073/pnas.95.18.10570. PMC 27935. PMID 9724744. 
  4. ^ Kim, Y. C., Grable, J. C., Love, R., Greene, P. J., Rosenberg, J. M. (1990). "Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing". Science 249 (4974): 1307–1309. doi:10.1126/science.2399465. PMID 2399465. 
  5. ^ http://www.neb.com/nebecomm/products/faqproductR0101.asp#1

External links

This page is based on a Wikipedia article. The text is available under the Creative Commons Attribution/Share-Alike License.

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Restriction endonuclease EcoRI Provide feedback

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Literature references

  1. Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM; , Science 1990;249:1307-1309.: Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing. PUBMED:2399465 EPMC:2399465

  2. Steczkiewicz K, Muszewska A, Knizewski L, Rychlewski L, Ginalski K;, Nucleic Acids Res. 2012;40:7016-7045.: Sequence, structure and functional diversity of PD-(D/E)XK phosphodiesterase superfamily. PUBMED:22638584 EPMC:22638584


External database links

This tab holds annotation information from the InterPro database.

InterPro entry IPR004221

There are four classes of restriction endonucleases: types I, II,III and IV. All types of enzymes recognise specific short DNA sequences and carry out the endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5'-phosphates. They differ in their recognition sequence, subunit composition, cleavage position, and cofactor requirements [PUBMED:15121719, PUBMED:12665693], as summarised below:

  • Type I enzymes (EC) cleave at sites remote from recognition site; require both ATP and S-adenosyl-L-methionine to function; multifunctional protein with both restriction and methylase (EC) activities.
  • Type II enzymes (EC) cleave within or at short specific distances from recognition site; most require magnesium; single function (restriction) enzymes independent of methylase.
  • Type III enzymes (EC) cleave at sites a short distance from recognition site; require ATP (but doesn't hydrolyse it); S-adenosyl-L-methionine stimulates reaction but is not required; exists as part of a complex with a modification methylase methylase (EC).
  • Type IV enzymes target methylated DNA.

Type II restriction endonucleases (EC) are components of prokaryotic DNA restriction-modification mechanisms that protect the organism against invading foreign DNA. These site-specific deoxyribonucleases catalyse the endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5'-phosphates. Of the 3000 restriction endonucleases that have been characterised, most are homodimeric or tetrameric enzymes that cleave target DNA at sequence-specific sites close to the recognition site. For homodimeric enzymes, the recognition site is usually a palindromic sequence 4-8 bp in length. Most enzymes require magnesium ions as a cofactor for catalysis. Although they can vary in their mode of recognition, many restriction endonucleases share a similar structural core comprising four beta-strands and one alpha-helix, as well as a similar mechanism of cleavage, suggesting a common ancestral origin [PUBMED:15770420]. However, there is still considerable diversity amongst restriction endonucleases [PUBMED:14576294, PUBMED:11827971]. The target site recognition process triggers large conformational changes of the enzyme and the target DNA, leading to the activation of the catalytic centres. Like other DNA binding proteins, restriction enzymes are capable of non-specific DNA binding as well, which is the prerequisite for efficient target site location by facilitated diffusion. Non-specific binding usually does not involve interactions with the bases but only with the DNA backbone [PUBMED:11557805].

This entry represents restriction endonucleases EcoRI, which requires magnesium as a cofactor. EcoRI recognises the DNA sequence GAATTC and cleaves after G-1 [PUBMED:11170385].

Gene Ontology

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Domain organisation

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Alignments

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Representative proteomes NCBI
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RP15
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RP35
(4)
RP55
(5)
RP75
(6)
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You can also download a FASTA format file containing the full-length sequences for all sequences in the full alignment.

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Curation and family details

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Seed source: Structural domain
Previous IDs: none
Type: Domain
Author: Griffiths-Jones SR
Number in seed: 3
Number in full: 52
Average length of the domain: 187.20 aa
Average identity of full alignment: 49 %
Average coverage of the sequence by the domain: 86.48 %

HMM information View help on HMM parameters

HMM build commands:
build method: hmmbuild -o /dev/null HMM SEED
search method: hmmsearch -Z 23193494 -E 1000 --cpu 4 HMM pfamseq
Model details:
Parameter Sequence Domain
Gathering cut-off 19.9 19.9
Trusted cut-off 20.3 20.7
Noise cut-off 18.6 19.4
Model length: 257
Family (HMM) version: 11
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Species distribution

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Interactions

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EcoRI

Structures

For those sequences which have a structure in the Protein DataBank, we use the mapping between UniProt, PDB and Pfam coordinate systems from the PDBe group, to allow us to map Pfam domains onto UniProt sequences and three-dimensional protein structures. The table below shows the structures on which the EcoRI domain has been found. There are 10 instances of this domain found in the PDB. Note that there may be multiple copies of the domain in a single PDB structure, since many structures contain multiple copies of the same protein seqence.

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