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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.

SUBMITTER: Pernstich C

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

REPOSITORIES: biostudies-literature

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Illuminating the reaction pathway of the FokI restriction endonuclease by fluorescence resonance energy transfer.

Pernstich Christian C Halford Stephen E SE

Nucleic acids research 20111012 3

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 resonanc ...[more]

PMID: 21993298

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Project description:Conjugated polydiacetylene (PDA) possessing stimuli-responsive properties has been intensively investigated for developing efficient sensors. We report here fluorescence resonance energy transfer (FRET) in liposomes synthesized using different molar ratios of dansyl-tagged diacetylene and diacetylene-carboxylic acid monomers. Photopolymerization of diacetylene resulted in cross-linked PDA liposomes. We used steady-state electronic absorption, emission, and fluorescence anisotropy (FA) analysis to characterize the thermal-induced FRET between dansyl fluorophores (donor) and PDA (acceptor). We found that the monomer ratio of acceptor to donor ( R ad) and length of linkers (functional part that connects dansyl fluorophores to the diacetylene group in the monomer) strongly affected FRET. For R ad = 10 000, the acceptor emission intensity was amplified by more than 18 times when the liposome solution was heated from 298 to 338 K. A decrease in R ad resulted in diminished acceptor emission amplification. This was primarily attributed to lower FRET efficiency between donors and acceptors and a higher background signal. We also found that the FRET amplification of PDA emissions after heating the solution was much higher when dansyl was linked to diacetylene through longer and flexible linkers than through shorter linkers. We attributed this to insertion of dansyl in the bilayer of the liposomes, which led to an increased dansyl quantum yield and a higher interaction of multiple acceptors with limited available donors. This was not the case for shorter and more rigid linkers where PDA amplification was much smaller. The present studies aim at enhancing our understanding of FRET between fluorophores and PDA-based conjugated liposomes. Furthermore, receptor tagged onto PDA liposomes can interact with ligands present on proteins, enzymes, and cells, which will produce emission sensing signal. Therefore, using the present approach, there exist opportunities for designing FRET-based highly sensitive and selective chemical and biochemical sensors.

Dataset Information

Illuminating the reaction pathway of the FokI restriction endonuclease by fluorescence resonance energy transfer.

Publications

Illuminating the reaction pathway of the FokI restriction endonuclease by fluorescence resonance energy transfer.

Similar Datasets

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