title: Time-resolved fluorescence studies of nucleotide flipping by restriction enzymes.
abstract: Restriction enzymes Ecl18kI, PspGI and EcoRII-C, specific for interrupted 5-bp target sequences, flip the central base pair of these sequences into their protein pockets to facilitate sequence recognition and adjust the DNA cleavage pattern. We have used time-resolved fluorescence spectroscopy of 2-aminopurine-labelled DNA in complex with each of these enzymes in solution to explore the nucleotide flipping mechanism and to obtain a detailed picture of the molecular environment of the extrahelical bases. We also report the first study of the 7-bp cutter, PfoI, whose recognition sequence (T/CCNGGA) overlaps with that of the Ecl18kI-type enzymes, and for which the crystal structure is unknown. The time-resolved fluorescence experiments reveal that PfoI also uses base flipping as part of its DNA recognition mechanism and that the extrahelical bases are captured by PfoI in binding pockets whose structures are quite different to those of the structurally characterized enzymes Ecl18kI, PspGI and EcoRII-C. The fluorescence decay parameters of all the enzyme-DNA complexes are interpreted to provide insight into the mechanisms used by these four restriction enzymes to flip and recognize bases and the relationship between nucleotide flipping and DNA cleavage.
authors.affiliation: Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, 3001 Heverlee, Belgium. robert.neely@chem.kuleuven.be
authors.forename: Robert K
authors.initials: RK
authors.lastname: Neely
authors.forename: Gintautas
authors.initials: G
authors.lastname: Tamulaitis
authors.forename: Kai
authors.initials: K
authors.lastname: Chen
authors.forename: Marta
authors.initials: M
authors.lastname: Kubala
authors.forename: Virginijus
authors.initials: V
authors.lastname: Siksnys
authors.forename: Anita C
authors.initials: AC
authors.lastname: Jones
mesh_terms: D004247:DNA / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005453:Fluorescence; D009711:Nucleotides / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D009711:Nucleotides; D004247:DNA; D004262:DNA Restriction Enzymes; C114636:endodeoxyribonuclease Ecl18kI; C046545:CCWGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific