Nucleotide flips determine the specificity of the Ecl18kI restriction endonuclease.

The EMBO Journal
Matthias BochtlerV 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.

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Citations

Mar 9, 2007·Nucleic Acids Research·Magdalena Kaus-DrobekMatthias Bochtler
Jul 10, 2007·Nucleic Acids Research·Gintautas TamulaitisVirginijus Siksnys
May 3, 2008·Nucleic Acids Research·Dalia DaujotyteSaulius Klimasauskas
Aug 23, 2008·Nucleic Acids Research·Michael A Carpenter, Ashok S Bhagwat
Sep 30, 2008·Nucleic Acids Research·Gintautas TamulaitisVirginijus Siksnys
Oct 3, 2008·Nucleic Acids Research·Roman H SzczepanowskiMatthias Bochtler
Sep 11, 2009·Nucleic Acids Research·Robert K NeelyAnita C Jones
Jun 24, 2010·Nucleic Acids Research·Mindaugas ZarembaVirginijus Siksnys
Sep 24, 2010·Nucleic Acids Research·Malgorzata FirczukMatthias Bochtler
Mar 1, 2012·Nucleic Acids Research·David A RuslingStephen E Halford
Apr 13, 2012·Nucleic Acids Research·Elena ManakovaVirginijus Siksnys
May 29, 2012·Nucleic Acids Research·Kamil SteczkiewiczKrzysztof Ginalski
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Dec 5, 2015·Nucleic Acids Research·Karolina MierzejewskaHonorata Czapinska
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May 10, 2021·Biochimica Et Biophysica Acta. General Subjects·Elena ManakovaGiedre Tamulaitiene
Mar 8, 2019·Journal of the American Chemical Society·Blaine J DowAlexander C Drohat

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