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Nucleotide flips determine the specificity of the Ecl 18kI restriction endonuclease
Author(s) -
Bochtler Matthias,
Szczepanowski Roman H,
Tamulaitis Gintautas,
Grazulis Saulius,
Czapinska Honorata,
Manakova Elena,
Siksnys Virginijus
Publication year - 2006
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1038/sj.emboj.7601096
Subject(s) - biology , restriction enzyme , molecular cell biology , genetics , library science , dna , microbiology and biotechnology , computer science
Restricion endonuclease Ecl 18kI is specific for the sequence /CCNGG and cleaves it before the outer C to generate 5 nt 5′‐overhangs. It has been suggested that Ecl 18kI is evolutionarily related to Ngo MIV, a 6‐bp cutter that cleaves the sequence G/CCGGC and leaves 4 nt 5′‐overhangs. Here, we report the crystal structure of the Ecl 18kI–DNA complex at 1.7 Å resolution and compare it with the known structure of the Ngo MIV–DNA complex. We find that Ecl 18kI 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 Ecl 18kI and Ngo MIV 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, Ecl 18kI is the first example of a restriction endonuclease that flips nucleotides to achieve specificity for its recognition site.