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PDBsum entry 2fqz

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protein dna_rna Protein-protein interface(s) links
Hydrolase/DNA PDB id
2fqz
Jmol
Contents
Protein chains
290 a.a. *
DNA/RNA
Waters ×555
* Residue conservation analysis
PDB id:
2fqz
Name: Hydrolase/DNA
Title: Metal-depleted ecl18ki in complex with uncleaved DNA
Structure: DNA strand 1. Chain: e, g. Engineered: yes. DNA strand 2. Chain: f, h. Engineered: yes. R.Ecl18ki. Chain: a, b, c, d. Synonym: restriction endonuclease.
Source: Synthetic: yes. Enterobacter cloacae. Organism_taxid: 550. Gene: ecl18kir. Expressed in: escherichia coli. Expression_system_taxid: 562.
Biol. unit: Tetramer (from PQS)
Resolution:
2.00Å     R-factor:   0.220     R-free:   0.260
Authors: M.Bochtler,R.H.Szczepanowski,G.Tamulaitis,S.Grazulis,H.Czapi E.Manakova,V.Siksnys
Key ref:
M.Bochtler et al. (2006). Nucleotide flips determine the specificity of the Ecl18kI restriction endonuclease. EMBO J, 25, 2219-2229. PubMed id: 16628220 DOI: 10.1038/sj.emboj.7601096
Date:
18-Jan-06     Release date:   20-Jun-06    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chains
Pfam   ArchSchema ?
O87963  (O87963_ENTCL) -  R.Ecl18kI (Restriction endonuclease)
Seq:
Struc:
305 a.a.
290 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 1 residue position (black cross)

 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     nucleic acid phosphodiester bond hydrolysis   3 terms 
  Biochemical function     DNA binding     3 terms  

 

 
DOI no: 10.1038/sj.emboj.7601096 EMBO J 25:2219-2229 (2006)
PubMed id: 16628220  
 
 
Nucleotide flips determine the specificity of the Ecl18kI restriction endonuclease.
M.Bochtler, R.H.Szczepanowski, G.Tamulaitis, S.Grazulis, H.Czapinska, E.Manakova, V.Siksnys.
 
  ABSTRACT  
 
Restricion endonuclease Ecl18kI is specific for the sequence /CCNGG and cleaves it before the outer C to generate 5 nt 5'-overhangs. It has been suggested that Ecl18kI is evolutionarily related to NgoMIV, a 6-bp cutter that cleaves the sequence G/CCGGC and leaves 4 nt 5'-overhangs. Here, we report the crystal structure of the Ecl18kI-DNA complex at 1.7 A resolution and compare it with the known structure of the NgoMIV-DNA complex. We find that Ecl18kI flips both central nucleotides within the CCNGG sequence and buries the extruded bases in pockets within the protein. Nucleotide flipping disrupts Watson-Crick base pairing, induces a kink in the DNA and shifts the DNA register by 1 bp, making the distances between scissile phosphates in the Ecl18kI and NgoMIV cocrystal structures nearly identical. Therefore, the two enzymes can use a conserved DNA recognition module, yet recognize different sequences, and form superimposable dimers, yet generate different cleavage patterns. Hence, Ecl18kI is the first example of a restriction endonuclease that flips nucleotides to achieve specificity for its recognition site.
 
  Selected figure(s)  
 
Figure 1.
Figure 1 Oligonucleotides used for cocrystallization of Ecl18kI (this work) and NgoMIV (Deibert et al, 2000). The recognition sequence is shown in bold letters, boxes indicate the cleavage patterns with 5 nt and 4 nt 5' overhangs, respectively.
Figure 5.
Figure 5 Comparison between Ecl18kI and NgoMIV. (A) Stereoview of the superimposed active sites of Ecl18kI and NgoMIV (shown in orange and green colors, respectively). Active site residues of NgoMIV Glu70, Asp140, Lys187 and Glu201 superimpose with Ecl18kI residues Glu125, Asp160, Lys182 and Glu195 demonstrated to be important in catalysis (Tamulaitis et al, 2002). Two Mg^2+-ions present in NgoMIV–DNA complex are shown as gray spheres, water molecules are not shown. (B) Stereoview of the conserved dimer interface between Ecl18kI and NgoMIV. The Ecl18kI dimer (orange) was superimposed with an equivalent pair of NgoMIV protomers (green). Large spheres indicate C -atoms of residues implicated in catalysis. Small spheres indicate C -atoms of residues that form hydrogen bonds with bases of the target sequence. The yellow labels on the left side indicate residues in Ecl18kI, the green labels on the right side mark the corresponding residues in NgoMIV.
 
  The above figures are reprinted by permission from Macmillan Publishers Ltd: EMBO J (2006, 25, 2219-2229) copyright 2006.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
21347256 D.Duzdevich, J.Li, J.Whang, H.Takahashi, K.Takeyasu, D.T.Dryden, A.J.Morton, and J.M.Edwardson (2011).
Unusual Structures Are Present in DNA Fragments Containing Super-Long Huntingtin CAG Repeats.
  PLoS One, 6, e17119.  
20861000 M.Firczuk, M.Wojciechowski, H.Czapinska, and M.Bochtler (2011).
DNA intercalation without flipping in the specific ThaI-DNA complex.
  Nucleic Acids Res, 39, 744-754.
PDB code: 3ndh
20541511 B.W.Shen, D.F.Heiter, S.H.Chan, H.Wang, S.Y.Xu, R.D.Morgan, G.G.Wilson, and B.L.Stoddard (2010).
Unusual target site disruption by the rare-cutting HNH restriction endonuclease PacI.
  Structure, 18, 734-743.
PDB codes: 3ldy 3m7k
20571089 M.Zaremba, A.Owsicka, G.Tamulaitis, G.Sasnauskas, L.S.Shlyakhtenko, A.Y.Lushnikov, Y.L.Lyubchenko, N.Laurens, B.van den Broek, G.J.Wuite, and V.Siksnys (2010).
DNA synapsis through transient tetramerization triggers cleavage by Ecl18kI restriction enzyme.
  Nucleic Acids Res, 38, 7142-7154.  
19298824 A.Alian, A.DeGiovanni, S.L.Griner, J.S.Finer-Moore, and R.M.Stroud (2009).
Crystal structure of an RluF-RNA complex: a base-pair rearrangement is the key to selectivity of RluF for U2604 of the ribosome.
  J Mol Biol, 388, 785-800.
PDB code: 3dh3
19729506 D.Golovenko, E.Manakova, G.Tamulaitiene, S.Grazulis, and V.Siksnys (2009).
Structural mechanisms for the 5'-CCWGG sequence recognition by the N- and C-terminal domains of EcoRII.
  Nucleic Acids Res, 37, 6613-6624.
PDB codes: 3hqf 3hqg
19740769 R.K.Neely, G.Tamulaitis, K.Chen, M.Kubala, V.Siksnys, and A.C.Jones (2009).
Time-resolved fluorescence studies of nucleotide flipping by restriction enzymes.
  Nucleic Acids Res, 37, 6859-6870.  
18400177 A.R.Lambert, D.Sussman, B.Shen, R.Maunus, J.Nix, J.Samuelson, S.Y.Xu, and B.L.Stoddard (2008).
Structures of the rare-cutting restriction endonuclease NotI reveal a unique metal binding fold involved in DNA binding.
  Structure, 16, 558-569.
PDB codes: 3bvq 3c25
18450817 D.Daujotyte, Z.Liutkeviciūte, G.Tamulaitis, and S.Klimasauskas (2008).
Chemical mapping of cytosines enzymatically flipped out of the DNA helix.
  Nucleic Acids Res, 36, e57.  
18522280 E.A.Fedotova, F.Ian, E.A.Kubareva, E.A.Romanova, A.S.Protsenko, M.B.Viriasov, T.Hianik, and T.S.Oretskaia (2008).
[Synthesis and characteristics of modified DNA fragments containing thymidine glycol residues]
  Bioorg Khim, 34, 236-244.  
18820295 G.Tamulaitis, M.Zaremba, R.H.Szczepanowski, M.Bochtler, and V.Siksnys (2008).
How PspGI, catalytic domain of EcoRII and Ecl18kI acquire specificities for different DNA targets.
  Nucleic Acids Res, 36, 6101-6108.  
18718929 M.A.Carpenter, and A.S.Bhagwat (2008).
DNA base flipping by both members of the PspGI restriction-modification system.
  Nucleic Acids Res, 36, 5417-5425.  
18829716 R.H.Szczepanowski, M.A.Carpenter, H.Czapinska, M.Zaremba, G.Tamulaitis, V.Siksnys, A.S.Bhagwat, and M.Bochtler (2008).
Central base pair flipping and discrimination by PspGI.
  Nucleic Acids Res, 36, 6109-6117.  
17617640 G.Tamulaitis, M.Zaremba, R.H.Szczepanowski, M.Bochtler, and V.Siksnys (2007).
Nucleotide flipping by restriction enzymes analyzed by 2-aminopurine steady-state fluorescence.
  Nucleic Acids Res, 35, 4792-4799.  
17344322 M.Kaus-Drobek, H.Czapinska, M.Sokołowska, G.Tamulaitis, R.H.Szczepanowski, C.Urbanke, V.Siksnys, and M.Bochtler (2007).
Restriction endonuclease MvaI is a monomer that recognizes its target sequence asymmetrically.
  Nucleic Acids Res, 35, 2035-2046.
PDB codes: 2oa9 2oaa
17392342 R.Sukackaite, A.Lagunavicius, K.Stankevicius, C.Urbanke, C.Venclovas, and V.Siksnys (2007).
Restriction endonuclease BpuJI specific for the 5'-CCCGT sequence is related to the archaeal Holliday junction resolvase family.
  Nucleic Acids Res, 35, 2377-2389.  
16893959 M.Carpenter, P.Divvela, V.Pingoud, J.Bujnicki, and A.S.Bhagwat (2006).
Sequence-dependent enhancement of hydrolytic deamination of cytosines in DNA by the restriction enzyme PspGI.
  Nucleic Acids Res, 34, 3762-3770.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB code is shown on the right.