Nucleotide flips determine the specificity of the Ecl18kI restriction endonuclease.
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.
Bochtler
Matthias
M
International Institute of Molecular and Cell Biology, Warsaw, Poland. MBochtler@iimcb.gov.pl
Szczepanowski
Roman H
RH
Tamulaitis
Gintautas
G
Grazulis
Saulius
S
Czapinska
Honorata
H
Manakova
Elena
E
Siksnys
Virginijus
V
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
D046911:Macromolecular Substances; D009711:Nucleotides; D004247:DNA; C114636:endodeoxyribonuclease Ecl18kI; D015252:Deoxyribonucleases, Type II Site-Specific