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Nucleotide flips determine the specificity of the Ecl18kI restriction endonuclease

Bochtler, Matthias, Szczepanowski, Roman H., Tamulaitis, Gintautas, Grazulis, Saulius, Czapinska, Honorata, Manakova, Elena and Siksnys, Virginijus 2006. Nucleotide flips determine the specificity of the Ecl18kI restriction endonuclease. The EMBO Journal 25 (10) , pp. 2219-2229. 10.1038/sj.emboj.7601096

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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 Å 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.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Chemistry
ISSN: 02614189
Last Modified: 19 Mar 2016 22:03
URI: http://orca.cf.ac.uk/id/eprint/1476

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