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 disen

SUBMITTER: Pernstich C 

PROVIDER: S-EPMC3273807 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature