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3  structures 73  species 0  interactions 84  sequences 3  architectures

Family: EcoRII-N (PF09217)

Summary: Restriction endonuclease EcoRII, N-terminal

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This is the Wikipedia entry entitled "R.EcoRII". More...

R.EcoRII Edit Wikipedia article

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 composed of 402 amino acids.[1]

Mode of action[edit]

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]

Cut diagram[edit]

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

Structure[edit]

Restriction endonuclease EcoRII, N-terminal
PDB 1na6 EBI.jpg
crystal structure of restriction endonuclease ecorii mutant r88a
Identifiers
Symbol EcoRII-N
Pfam PF09217
Pfam clan CL0405
InterPro IPR015300
SCOP 1na6
SUPERFAMILY 1na6
EcoRII C terminal
PDB 1na6 EBI.jpg
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.

Effector-binding domain[edit]

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:

Catalytic domain[edit]

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).

Autoinhibition/activation mechanism[edit]

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[edit]

External links[edit]

  • EcoRII in Restriction Enzyme Database REBASE

References[edit]

  1. ^ Richard J. Roberts. "EcoRII". REBASE - The Restriction Enzyme Database. Retrieved 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. PMC 152790. 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. PMC 535885. PMID 15548737. PDF
  8. ^ Richard J. Roberts. "BfiI". REBASE - The Restriction Enzyme Database. Retrieved 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. PMC 1266039. 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. PMC 1874628. PMID 17369272.  PDF


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Restriction endonuclease EcoRII, N-terminal Provide feedback

The N-terminal effector-binding domain of the Restriction Endonuclease EcoRII has a DNA recognition fold, allowing for binding to 5'-CCWGG sequences. It assumes a structure composed of an eight-stranded beta-sheet with the strands in the order of b2, b5, b4, b3, b7, b6, b1 and b8. They are mostly antiparallel to each other except that b3 is parallel to b7. Alternatively, it may also be viewed as consisting of two mini beta-sheets of four antiparallel beta-strands, sheet I from beta-strands b2, b5, b4, b3 and sheet II from strands b7, b6, b1, b8, folded into an open mixed beta-barrel with a novel topology. Sheet I has a simple Greek key motif while sheet II does not [1].

Literature references

  1. Zhou XE, Wang Y, Reuter M, Mucke M, Kruger DH, Meehan EJ, Chen L; , J Mol Biol. 2004;335:307-319.: Crystal structure of type IIE restriction endonuclease EcoRII reveals an autoinhibition mechanism by a novel effector-binding fold. PUBMED:14659759 EPMC:14659759


External database links

This tab holds annotation information from the InterPro database.

InterPro entry IPR023372

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 the N-terminal effector-binding domain of the type II restriction endonuclease EcoRII, which has a DNA recognition fold, allowing for binding to 5'-CCWGG sequences. It assumes a structure composed of an eight-stranded beta-sheet with the strands in the order of b2, b5, b4, b3, b7, b6, b1 and b8. They are mostly antiparallel to each other except that b3 is parallel to b7. Alternatively, it may also be viewed as consisting of two mini beta-sheets of four antiparallel beta-strands, sheet I from beta-strands b2, b5, b4, b3 and sheet II from strands b7, b6, b1, b8, folded into an open mixed beta-barrel with a novel topology. Sheet I has a simple Greek key motif while sheet II does not [PUBMED:14659759].

The domain represented by this entry is only found in bacterial proteins.

Domain organisation

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Pfam Clan

This family is a member of clan DNA_b-psBarrel (CL0405), which has the following description:

Superfamily consists of type II restriction endonuclease effector (N-term) domain and plant B3 DNA binding domain families.

The clan contains the following 3 members:

B3 DUF313 EcoRII-N

Alignments

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RP55
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(6)
Full
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Representative proteomes NCBI
(59)
Meta
(2)
RP15
(4)
RP35
(6)
RP55
(8)
RP75
(10)
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Curation and family details

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Curation View help on the curation process

Seed source: pdb_1na6
Previous IDs: none
Type: Domain
Author: Sammut SJ
Number in seed: 6
Number in full: 84
Average length of the domain: 147.10 aa
Average identity of full alignment: 46 %
Average coverage of the sequence by the domain: 39.22 %

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 25.0 25.0
Trusted cut-off 34.1 33.6
Noise cut-off 24.4 23.6
Model length: 156
Family (HMM) version: 5
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Species distribution

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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 EcoRII-N domain has been found. There are 3 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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