Restriction enzyme Ecl18kI-DNA complex
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.
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.
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. The DNA conformation in Ecl18kI. The arrowheads(△) show the central extruded nucleotides.
Protein Data Bank (PDB)
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.
author: Daisuke Ino