Restriction endonuclease (REase) EcoRII (pronounced "eco R two") 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. [1]
EcoRII is a bacterial Type IIE [2] REase that interacts with two [3] or three [4] copies of the pseudopalindromic DNA recognition sequence 5'-CCWGG-3' (W = A or T), one being the actual target of cleavage, the other(s) serving as the allosteric activator(s). EcoRII cuts the target DNA sequence CCWGG, generating sticky ends. [5]
| Recognition site | Cut results |
5' NNCCWGGNN 3' NNGGWCCNN | 5' NN CCWGGNN 3' NNGGWCC NN |
| Restriction endonuclease EcoRII, N-terminal | |||||||||
|---|---|---|---|---|---|---|---|---|---|
 crystal structure of restriction endonuclease ecorii mutant r88a | |||||||||
| Identifiers | |||||||||
| Symbol | EcoRII-N | ||||||||
| Pfam | PF09217 | ||||||||
| Pfam clan | CL0405 | ||||||||
| InterPro | IPR015300 | ||||||||
| SCOP2 | 1na6 / SCOPe / SUPFAM | ||||||||
| |||||||||
| EcoRII C terminal | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| crystal structure of restriction endonuclease ecorii mutant r88a | |||||||||
| Identifiers | |||||||||
| Symbol | EcoRII-C | ||||||||
| Pfam | PF09019 | ||||||||
| Pfam clan | CL0236 | ||||||||
| InterPro | IPR015109 | ||||||||
| |||||||||
The apo crystal structure of EcoRII mutant R88A ( PDB: 1NA6 ) [6] has been solved at 2.1 Å resolution. The EcoRII monomer has two domains, N-terminal and C-terminal, linked through a hinge loop.
The N-terminal effector-binding domain has an archetypal DNA-binding pseudobarrel fold ( SCOP 101936 ) with a prominent cleft. Structural superposition showed it is evolutionarily related to:
The C-terminal catalytic domain has a typical [10] restriction endonuclease-like fold ( SCOP 52979 ) and belongs to the large (more than 30 members) restriction endonuclease superfamily ( SCOP 52980 ).
Structure-based sequence alignment and site-directed mutagenesis identified the putative PD..D/EXK active sites of the EcoRII catalytic domain dimer that in apo structure are spatially blocked by the N-terminal domains. [6]
A restriction enzyme, restriction endonuclease, or restrictase 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.
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.
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.
Micrococcal nuclease is an endo-exonuclease that preferentially digests single-stranded nucleic acids. The rate of cleavage is 30 times greater at the 5' side of A or T than at G or C and results in the production of mononucleotides and oligonucleotides with terminal 3'-phosphates. The enzyme is also active against double-stranded DNA and RNA and all sequences will be ultimately cleaved.
A DNA-binding domain (DBD) is an independently folded protein domain that contains at least one structural motif that recognizes double- or single-stranded DNA. A DBD can recognize a specific DNA sequence or have a general affinity to DNA. Some DNA-binding domains may also include nucleic acids in their folded structure.
Exonuclease III (ExoIII) is an enzyme that belongs to the exonuclease family. ExoIII catalyzes the stepwise removal of mononucleotides from 3´-hydroxyl termini of double-stranded DNA. A limited number of nucleotides are removed during each binding event, resulting in coordinated progressive deletions within the population of DNA molecules.
DNA adenine methylase, (Dam methylase) is an enzyme that adds a methyl group to the adenine of the sequence 5'-GATC-3' in newly synthesized DNA. Immediately after DNA synthesis, the daughter strand remains unmethylated for a short time. It is an orphan methyltransferase that is not part of a restriction-modification system and regulates gene expression. This enzyme catalyses the following chemical reaction
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-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, without further sequence specificity, the first strand 9 nucleotides downstream and the second strand 13 nucleotides upstream of the nearest nucleotide of the recognition site.
EcoRV is a type II restriction endonuclease isolated from certain strains of Escherichia coli. It has the alternative name Eco32I.
Flap endonucleases are a class of nucleolytic enzymes that act as both 5'-3' exonucleases and structure-specific endonucleases on specialised DNA structures that occur during the biological processes of DNA replication, DNA repair, and DNA recombination. Flap endonucleases have been identified in eukaryotes, prokaryotes, archaea, and some viruses. Organisms can have more than one FEN homologue; this redundancy may give an indication of the importance of these enzymes. In prokaryotes, the FEN enzyme is found as an N-terminal domain of DNA polymerase I, but some prokaryotes appear to encode a second homologue.
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 a kind of Endonuclease that catalyzes the degradation nucleotides in DsDNA by attacking the 5'-terminal end.
The degradosome is a multiprotein complex present in most bacteria that is involved in the processing of ribosomal RNA and the degradation of messenger RNA and is regulated by Non-coding RNA. It contains the proteins RNA helicase B, RNase E and Polynucleotide phosphorylase.
Acid-Induced Arginine Decarboxylase (AdiA), also commonly referred to as arginine decarboxylase, is an enzyme responsible for catalyzing the conversion of L-arginine into agmatine and carbon dioxide. The process consumes a proton in the decarboxylation and employs a pyridoxal-5'-phosphate (PLP) cofactor, similar to other enzymes involved in amino acid metabolism, such as ornithine decarboxylase and glutamine decarboxylase. It is found in bacteria and virus, though most research has so far focused on forms of the enzyme in bacteria. During the AdiA catalyzed decarboxylation of arginine, the necessary proton is consumed from the cell cytoplasm which helps to prevent the over-accumulation of protons inside the cell and serves to increase the intracellular pH. Arginine decarboxylase is part of an enzymatic system in Escherichia coli, Salmonella Typhimurium, and methane-producing bacteria Methanococcus jannaschii that makes these organisms acid resistant and allows them to survive under highly acidic medium.
The B3 DNA binding domain (DBD) is a highly conserved domain found exclusively in transcription factors combined with other domains. It consists of 100-120 residues, includes seven beta strands and two alpha helices that form a DNA-binding pseudobarrel protein fold ; it interacts with the major groove of DNA.
PstI is a type II restriction endonuclease isolated from the Gram negative species, Providencia stuartii.
EcoRI 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. 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.
