R.EcoRII

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Eco RII dimer based on PDB ID 1NA6
Eco RII dimer based on PDB ID 1NA6

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 comprised of 402 amino acids.[1]

Contents

[edit] 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 cut target DNA sequence CCWGG generating sticky ends.[5]

[edit] Cut diagram

Recognition site Cut results 
5' NNCCWGGNN
3' NNGGWCCNN 

5' NN  CCWGGNN
3' NNGGWCC  NN

[edit] Structure

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.

[edit] Effector-binding domain

The N-terminal effector-binding domain has a archetypal DNA-binding pseudobarrel fold (SCOP 101936) with a prominent cleft. Structural superposition showed it is evolutionarily related to:

[edit] Catalytic domain

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).

[edit] 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]

[edit] See also

[edit] External links

  • EcoRII in Restriction Enzyme Database REBASE

[edit] References

  1. ^ Richard J. Roberts. EcoRII. REBASE - The Restriction Enzyme Database. Retrieved on 2008-03-23.
  2. ^ Roberts RJ, Belfort M, Bestor T, et al (2003). "A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes". Nucleic Acids Res. 31 (7): 1805-12. doi:10.1093/nar/gkg274. PMID 12654995.  PDF
  3. ^ Mücke M, Lurz R, Mackeldanz P, Behlke J, Krüger DH, Reuter M (2000). "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. doi:10.1074/jbc.M003904200. PMID 10903314. PDF
  4. ^ Shlyakhtenko LS, Gilmore J, Portillo A, Tamulaitis G, Siksnys V, Lyubchenko YL (2007). "Direct visualization of the EcoRII-DNA triple synaptic complex by atomic force microscopy". Biochemistry 46 (39): 11128-36. doi:10.1021/bi701123u. PMID 17845057. 
  5. ^ Griffiths, Anthony J. F. (1999). An Introduction to genetic analysis. San Francisco: W.H. Freeman. ISBN 0-7167-3520-2. 
  6. ^ a b Zhou XE, Wang Y, Reuter M, Mücke M, Krüger DH, Meehan EJ, Chen L (2004). "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. doi:10.1016/j.jmb.2003.10.030. PMID 14659759. 
  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 (2004). "Solution structure of the B3 DNA binding domain of the Arabidopsis cold-responsive transcription factor RAV1". Plant Cell 16 (12): 3448-59. doi:10.1105/tpc.104.026112. PMID 15548737. PDF
  8. ^ Richard J. Roberts. BfiI. REBASE - The Restriction Enzyme Database. Retrieved on 2008-03-23.
  9. ^ Grazulis S, Manakova E, Roessle M, Bochtler M, Tamulaitiene G, Huber R, Siksnys V (2005). "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. doi:10.1073/pnas.0507949102. PMID 16247004.  PDF
  10. ^ Niv MY, Ripoll DR, Vila JA, Liwo A, Vanamee ES, Aggarwal AK, Weinstein H, Scheraga HA (2007). "Topology of Type II REases revisited; structural classes and the common conserved core". NAR 35 (7): 2227-37. doi:10.1093/nar/gkm045. PMID 17369272.  PDF
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