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PDB:2FQZ

Protein Name

Restriction enzyme Ecl18kI-DNA complex

Species

Enterobacter cloaceae

Biological Context

Restriction enzymes cleave the target DNA with specific sequence, and are essential for current genetic engineering. Many target sequences are palindromic with even number of base pairs, and are cleaved to generate either even number overhangs or blunt ends(fig1 A), while some target sequence are pseudplindromic with odd number of base pairs, and are cleaved to generate either odd number overhangs(fig1B). In particular, the protein described here, Ecl18kI, recognizes a pseudpalindromic sequence.

fig1e

fig1. (A) An example of palindromic sequence(restriction enzyme NgoM IV recognition sequence)and (B) An example of pseudpalindromic sequence(restriction enzyme Ecl18kI recognition sequence). Recognition sequnece is expressed as bold letters and the number above the sequence is the position from the center. The line shows the cleveage site.

Structure Description

2fqz2fqz_x2fqz_y

In the crystal of restriction enzyme Ecl18kI-DNA complex, an unit contains four Ecl18kI monomer and two DNAs, and two Ecl18kI dimers with a DNA lines asymmetrically to form tetramer (although whether this tetrameric structure is a physiological form or an artifact by crystal packing is unknown). The most characteristic point in this structure is the DNA conformation. As described in fig2, the nucleotides located at the center of the target sequence(in this case “A” and “T”) are flipped out, due to the disruption of the hydrogen bonds. In this state, Ecl18kI only recognize the palindromic sequence (CCGG) skipping the central nucleotide of the pseudpalindromic sequence. Comparison between the active site of Ecl18kI and that of NgoMIV which is a restriction enzyme cleaving GCCGGC palindromic sequence revealed their structural similarity, despite their low sequence homology. Flipping of the central nucleotide in the pseudpalindromic sequence is the key to determine the specificity of the Ecl18kI.

fig2

fig2. The DNA conformation in Ecl18kI. The arrowheads(△) show the central extruded nucleotides.

Protein Data Bank (PDB)

References

Source

Bochtler, M. Szczepanowski, R.H. Tamulaitis, G. Grazulis, S. Czapinska, H. Manakova, E. Siksnys, V.; "Nucleotide flips determine the specificity of the Ecl18kI restriction endonuclease"; Embo J.; (2006) 25:2219-2229 PubMed:16628220.

Others

author: Daisuke Ino


Japanese version:PDB:2FQZ