EcoRI

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EcoRI
Ecor1 2ckq.png
EcoRI crystal structure. Dimer bound to DNA (PDB 1ckq)
Identifiers
SymbolEcoRI
Pfam PF02963
InterPro IPR004221
SCOP2 1na6 / SCOPe / SUPFAM
CDD 79lll
Available protein structures:
Pfam   structures / ECOD  
PDB RCSB PDB; PDBe; PDBj
PDBsum structure summary

EcoRI (pronounced "eco R one") is a restriction endonuclease enzyme isolated from species E. coli. It is a restriction enzyme that cleaves DNA double helices into fragments at specific sites, and is also a part of the restriction modification system. [1] The Eco part of the enzyme's name originates from the species from which it was isolated - "E" denotes generic name which is "Escherichia" and "co" denotes species name, "coli" - while the R represents the particular strain, in this case RY13, and the I denotes that it was the first enzyme isolated from this strain.[ citation needed ]

Contents

In molecular biology it is used as a restriction enzyme. EcoRI creates 4 nucleotide sticky ends with 5' end overhangs of AATT. The nucleic acid recognition sequence where the enzyme cuts is G↓AATTC, which has a palindromic complementary sequence of CTTAA↓G. [2] Other restriction enzymes, depending on their cut sites, can also leave 3' overhangs or blunt ends with no overhangs.

History

EcoRI is an example of type II restriction enzymes which now has more the 300 enzymes with more than 200 different sequence-specificities, which has transformed molecular biology and medicine. [3]

EcoRI, discovered in 1970, was isolated by PhD student Robert Yoshimori who investigated clinical E. coli isolates that contained restriction systems presented on its plasmids. [3] The purified isolates became known as EcoRI that is used to cleave G’AATTC. [2]

Structure

Primary structure

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

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. [4] [5]

EcoRI recognition site with cutting pattern indicated by a green line EcoRI restriction enzyme recognition site.svg
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. [4] In the complex, two α-helices from each subunit come together to form a four-helix bundle. [6] 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. [7]

Uses

Restriction enzymes 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 sticky ends make the ligation reaction more efficient. [8] One example of this use is in recombinant DNA production, when joining donor and vector DNA. [9] 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. [10]

See also

Related Research Articles

A restriction enzyme, restriction endonuclease, REase, ENase orrestrictase is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class of the broader endonuclease group of enzymes. Restriction enzymes are commonly classified into five types, which differ in their structure and whether they cut their DNA substrate at their recognition site, or if the recognition and cleavage sites are separate from one another. To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone of the DNA double helix.

<span class="mw-page-title-main">Nuclease</span> Class of enzymes which cleave nucleic acids

In biochemistry, a nuclease is an enzyme capable of cleaving the phosphodiester bonds between nucleotides of nucleic acids. Nucleases variously effect single and double stranded breaks in their target molecules. In living organisms, they are essential machinery for many aspects of DNA repair. Defects in certain nucleases can cause genetic instability or immunodeficiency. Nucleases are also extensively used in molecular cloning.

In molecular biology, endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain. Some, such as deoxyribonuclease I, cut DNA relatively nonspecifically, while many, typically called restriction endonucleases or restriction enzymes, cleave only at very specific nucleotide sequences. Endonucleases differ from exonucleases, which cleave the ends of recognition sequences instead of the middle (endo) portion. Some enzymes known as "exo-endonucleases", however, are not limited to either nuclease function, displaying qualities that are both endo- and exo-like. Evidence suggests that endonuclease activity experiences a lag compared to exonuclease activity.

Restriction sites, or restriction recognition sites, are located on a DNA molecule containing specific sequences of nucleotides, which are recognized by restriction enzymes. These are generally palindromic sequences, and a particular restriction enzyme may cut the sequence between two nucleotides within its recognition site, or somewhere nearby.

<i>Nla</i>III Enzyme

NlaIII is a type II restriction enzyme isolated from Neisseria lactamica. As part of the restriction modification system, NlaIII is able to prevent foreign DNA from integrating into the host genome by cutting double stranded DNA into fragments at specific sequences. This results in further degradation of the fragmented foreign DNA and prevents it from infecting the host genome.

<i>Fok</i>I Restriction enzyme

The restriction endonuclease Fok1, naturally found in Flavobacterium okeanokoites, is a bacterial type IIS restriction endonuclease consisting of an N-terminal DNA-binding domain and a non sequence-specific DNA cleavage domain at the C-terminal. Once the protein is bound to duplex DNA via its DNA-binding domain at the 5'-GGATG-3' recognition site, the DNA cleavage domain is activated and cleaves the DNA at two locations, regardless of the nucleotide sequence at the cut site. The DNA is cut 9 nucleotides downstream of the motif on the forward strand, and 13 nucleotides downstream of the motif on the reverse strand, producing two sticky ends with 4-bp overhangs.

<i>Hin</i>dIII Enzyme

HindIII (pronounced "Hin D Three") is a type II site-specific deoxyribonuclease restriction enzyme isolated from Haemophilus influenzae that cleaves the DNA palindromic sequence AAGCTT in the presence of the cofactor Mg2+ via hydrolysis.

<i>Eco</i>RV Restriction enzyme

EcoRV is a type II restriction endonuclease isolated from certain strains of Escherichia coli. It has the alternative name Eco32I.

<span class="mw-page-title-main">Homing endonuclease</span> Type of enzyme

The homing endonucleases are a collection of endonucleases encoded either as freestanding genes within introns, as fusions with host proteins, or as self-splicing inteins. They catalyze the hydrolysis of genomic DNA within the cells that synthesize them, but do so at very few, or even singular, locations. Repair of the hydrolyzed DNA by the host cell frequently results in the gene encoding the homing endonuclease having been copied into the cleavage site, hence the term 'homing' to describe the movement of these genes. Homing endonucleases can thereby transmit their genes horizontally within a host population, increasing their allele frequency at greater than Mendelian rates.

Deoxyribonuclease IV (phage-T4-induced) is catalyzes the degradation nucleotides in DsDNA by attacking the 5'-terminal end.

<i>Bgl</i>II Restriction enzyme

BglII is a type II restriction endonuclease isolated from certain strains of Bacillus globigii.

<span class="mw-page-title-main">R.EcoRII</span> Restriction enzyme

Restriction endonuclease (REase) EcoRII is an enzyme of restriction modification system (RM) naturally found in Escherichia coli, a Gram-negative bacteria. Its molecular mass is 45.2 kDa, being composed of 402 amino acids.

PstI is a type II restriction endonuclease isolated from the Gram negative species, Providencia stuartii.

<span class="mw-page-title-main">Molecular cloning</span> Set of methods in molecular biology

Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word cloning refers to the fact that the method involves the replication of one molecule to produce a population of cells with identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA. Molecular cloning methods are central to many contemporary areas of modern biology and medicine.

<span class="mw-page-title-main">Ligation (molecular biology)</span> Technique for joining nucleic acid fragments

Ligation is the joining of two nucleic acid fragments through the action of an enzyme. It is an essential laboratory procedure in the molecular cloning of DNA, whereby DNA fragments are joined to create recombinant DNA molecules (such as when a foreign DNA fragment is inserted into a plasmid). The ends of DNA fragments are joined by the formation of phosphodiester bonds between the 3'-hydroxyl of one DNA terminus with the 5'-phosphoryl of another. RNA may also be ligated similarly. A co-factor is generally involved in the reaction, and this is usually ATP or NAD+. Eukaryotic cells ligases belong to ATP type, and NAD+ - dependent are found in bacteria (e.g. E. coli).

References

  1. Halford, S. E.; Johnson, N. P. (1980-11-01). "The EcoRI restriction endonuclease with bacteriophage lambda DNA. Equilibrium binding studies". The Biochemical Journal. 191 (2): 593–604. doi:10.1042/bj1910593. ISSN   0264-6021. PMC   1162251 . PMID   6263250.
  2. 1 2 Nevinsky, Georgy A. (2021-01-29). "How Enzymes, Proteins, and Antibodies Recognize Extended DNAs; General Regularities". International Journal of Molecular Sciences. 22 (3): 1369. doi: 10.3390/ijms22031369 . ISSN   1422-0067. PMC   7866405 . PMID   33573045.
  3. 1 2 Loenen, Wil A. M.; Dryden, David T. F.; Raleigh, Elisabeth A.; Wilson, Geoffrey G.; Murray, Noreen E. (January 2014). "Highlights of the DNA cutters: a short history of the restriction enzymes". Nucleic Acids Research. 42 (1): 3–19. doi:10.1093/nar/gkt990. ISSN   1362-4962. PMC   3874209 . PMID   24141096.
  4. 1 2 Pingoud A, Jeltsch A (September 2001). "Structure and function of type II restriction endonucleases". Nucleic Acids Research. 29 (18): 3705–27. doi:10.1093/nar/29.18.3705. PMC   55916 . PMID   11557805.
  5. Kurpiewski MR, Engler LE, Wozniak LA, Kobylanska A, Koziolkiewicz M, Stec WJ, Jen-Jacobson L (October 2004). "Mechanisms of coupling between DNA recognition specificity and catalysis in EcoRI endonuclease". Structure. 12 (10): 1775–88. doi: 10.1016/j.str.2004.07.016 . PMID   15458627.
  6. Bitinaite J, Wah DA, Aggarwal AK, Schildkraut I (September 1998). "FokI dimerization is required for DNA cleavage". Proceedings of the National Academy of Sciences of the United States of America. 95 (18): 10570–5. Bibcode:1998PNAS...9510570B. doi: 10.1073/pnas.95.18.10570 . PMC   27935 . PMID   9724744.
  7. Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM (September 1990). "Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing". Science. 249 (4974): 1307–9. Bibcode:1990Sci...249.1307K. doi:10.1126/science.2399465. PMID   2399465.
  8. Gao, T; Konomura, S; May, C; Nich, C (April 2015). "Increasing Overhang GC-Content Increases Sticky-End Ligation Efficiency" (PDF). Journal of Experimental Microbiology and Immunology.
  9. Griffiths, Anthony JF; Miller, Jeffrey H.; Suzuki, David T.; Lewontin, Richard C.; Gelbart, William M. (2000). "Making recombinant DNA". An Introduction to Genetic Analysis. 7th Edition.
  10. "FAQs for EcoRI, Restriction Endonucleases, NEB". Archived from the original on 2012-10-15. Retrieved 2010-01-21.