title: Illuminating the reaction pathway of the FokI restriction endonuclease by fluorescence resonance energy transfer.
abstract: The FokI restriction endonuclease is a monomeric protein that recognizes an asymmetric sequence and cleaves both DNA strands at fixed loci downstream of the site. Its single active site is positioned initially near the recognition sequence, distant from its downstream target 13 nucleotides away. Moreover, to cut both strands, it has to recruit a second monomer to give an assembly with two active sites. Here, the individual steps in the FokI reaction pathway were examined by fluorescence resonance energy transfer (FRET). To monitor DNA binding and domain motion, a fluorescence donor was attached to the DNA, either downstream or upstream of the recognition site, and an acceptor placed on the catalytic domain of the protein. A FokI variant incapable of dimerization was also employed, to disentangle the signal due to domain motion from that due to protein association. Dimerization was monitored separately by using two samples of FokI labelled with donor and acceptor, respectively. The stopped-flow studies revealed a complete reaction pathway for FokI, both the sequence of events and the kinetics of each individual step.
authors: The DNA-proteins Interaction Unit, School of Biochemistry, University of Bristol, University Walk, Bristol, BS8 1TD, UK.
authors: Christian
authors: C
authors: Pernstich
authors: Stephen E
authors: SE
authors: Halford
mesh_terms: D004247:DNA / Q000737:chemistry / Q000378:metabolism; D053837:DNA Cleavage; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D017434:Protein Structure, Tertiary
chemical_list: D004247:DNA; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific