R.EcoRII explained

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]

Mode of action

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]

Cut diagram

Recognition site	Cut results
5' NNCCWGGNN 3' NNGGWCCNN	5' NN CCWGGNN 3' NNGGWCC NN

Structure

Symbol:	EcoRII-N
Restriction endonuclease EcoRII, N-terminal
Pfam:	PF09217
Pfam Clan:	CL0405
Interpro:	IPR015300
Scop:	1na6

Symbol:	EcoRII-C
EcoRII C terminal
Pfam:	PF09019
Pfam Clan:	CL0236
Interpro:	IPR015109

The apo crystal structure of EcoRII mutant R88A ^[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.

Effector-binding domain

The N-terminal effector-binding domain has an archetypal DNA-binding pseudobarrel fold with a prominent cleft. Structural superposition showed it is evolutionarily related to:

• B3 DNA binding domain from the transcription factors in higher plants ^[7]
• C-terminal domain of restriction endonuclease BfiI^{[8] [9]}

Catalytic domain

The C-terminal catalytic domain has a typical^[10] restriction endonuclease-like fold and belongs to the large (more than 30 members) restriction endonuclease superfamily .

Autoinhibition/activation mechanism

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]

See also

• EcoRI, another nuclease enzyme from Escherichia coli.
• EcoRV, another nuclease enzyme from Escherichia coli.
• B3 DNA binding domain from higher plants is evolutionary related to EcoRII

• FokI, another nuclease enzyme from Flavobacterium okeanokoites

External links

• EcoRII in Restriction Enzyme Database REBASE

Notes and References

1. Web site: EcoRII . 2008-03-23 . Richard J. Roberts . REBASE - The Restriction Enzyme Database .
2. Roberts RJ, Belfort M, Bestor T, etal . A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes . Nucleic Acids Res. . 31 . 7 . 1805–12 . 2003 . 12654995 . 10.1093/nar/gkg274 . 152790 . PDF
3. Mücke M, Lurz R, Mackeldanz P, Behlke J, Krüger DH, Reuter M . Imaging DNA loops induced by restriction endonuclease EcoRII. A single amino acid substitution uncouples target recognition from cooperative DNA interaction and cleavage . J. Biol. Chem. . 275 . 39 . 30631–7 . 2000 . 10903314 . 10.1074/jbc.M003904200 . free . PDF
4. Shlyakhtenko LS, Gilmore J, Portillo A, Tamulaitis G, Siksnys V, Lyubchenko YL . 27800123 . Direct visualization of the EcoRII-DNA triple synaptic complex by atomic force microscopy . Biochemistry . 46 . 39 . 11128–36 . 2007 . 17845057 . 10.1021/bi701123u .
5. Book: Griffiths, Anthony J. F. . An Introduction to genetic analysis . W.H. Freeman . San Francisco . 1999 . 978-0-7167-3520-5 .
6. Zhou XE, Wang Y, Reuter M, Mücke M, Krüger DH, Meehan EJ, Chen L . Crystal structure of type IIE restriction endonuclease EcoRII reveals an autoinhibition mechanism by a novel effector-binding fold . J. Mol. Biol. . 335 . 1 . 307–19 . 2004 . 14659759 . 10.1016/j.jmb.2003.10.030 .
7. Yamasaki K, Kigawa T, Inoue M, Tateno M, Yamasaki T, Yabuki T, Aoki M, Seki E, Matsuda T, Tomo Y, Hayami N, Terada T, Shirouzu M, Osanai T, Tanaka A, Seki M, Shinozaki K, Yokoyama S . Solution structure of the B3 DNA binding domain of the Arabidopsis cold-responsive transcription factor RAV1 . Plant Cell . 16 . 12 . 3448–59 . 2004 . 15548737 . 10.1105/tpc.104.026112 . 535885 . PDF
8. Web site: BfiI . 2008-03-23 . Richard J. Roberts . REBASE - The Restriction Enzyme Database .
9. Grazulis S, Manakova E, Roessle M, Bochtler M, Tamulaitiene G, Huber R, Siksnys V . Structure of the metal-independent restriction enzyme BfiI reveals fusion of a specific DNA-binding domain with a nonspecific nuclease . Proc. Natl. Acad. Sci. U.S.A. . 102 . 44 . 15797–802 . 2005 . 16247004 . 10.1073/pnas.0507949102 . 1266039 . 2005PNAS..10215797G . free . PDF
10. Niv MY, Ripoll DR, Vila JA, Liwo A, Vanamee ES, Aggarwal AK, Weinstein H, Scheraga HA . Topology of Type II REases revisited; structural classes and the common conserved core . Nucleic Acids Research . 35 . 7 . 2227–37 . 2007 . 17369272 . 10.1093/nar/gkm045 . 1874628.